KR20130086386A - Biosynthetically generated pyrroline-carboxy-lysine and site specific protein modifications via chemical derivatization of pyrroline-carboxy-lysine and pyrrolysine residues - Google Patents

Abstract

Disclosed herein are pyrroline-carboxy-lysine (PCL), a biosynthetically produced natural amino acid and a pyrrolysine analog, and a method for biosynthetically producing PCL. Also disclosed herein are proteins, polypeptides and peptides having PCL incorporated therein, and methods of incorporating PCL into such proteins, polypeptides and peptides. Also disclosed herein are site specific derivatization of proteins, polypeptides and peptides having PCL or pyrrolysine incorporated therein. Also disclosed herein are crosslinking of proteins, polypeptides and peptides having PCL or pyrrolysine incorporated therein.

Description

Biosynthetically generated pyrroline-carboxy-lysine, and site-specific protein modification through chemical derivatization of pyrroline-carboxy-lysine and pyrrolysine residues {BIOSYNTHETICALLY GENERATED PYRROLINE-CARBOXY-LYSINE AND SITE SPECIFIC PROTEIN MODIFICATIONS VIA CHEMICAL DERIVATIZATION OF PYRROLINE-CARBOXY-LYSINE AND PYRROLYSINE RESIDUES}

<Cross reference to related applications>

This application claims the benefit of priority to U.S. Provisional Patent Application No. 61/108,434, filed Oct. 24, 2008 under 35 U.S.C. §119(e). The disclosure of the priority application is incorporated herein by reference in its entirety for all purposes.

<Technical field>

The present invention relates to the selective introduction of genetically encoded amino acids into proteins. The invention also relates to the chemical derivatization of these amino acids.

The methanogenic archaea methylamine methyltransferase of the family Methanosarcina naturally contains pyrrolysine (PYL). Pyrrolysine is a lysine analog that is incorporated concurrently with translation at the in-frame UAG codon within each mRNA, which is considered the 22nd natural amino acid.

Provided herein are proteins and/or polypeptides having one or more PCLs incorporated therein, wherein the PCL is produced biosynthetically and incorporated into the protein and/or polypeptide. Also provided herein are proteins and/or polypeptides having at least one pyrrolysine (PYL) incorporated therein, wherein PYL is produced biosynthetically and incorporated into the protein and/or polypeptide. Also provided herein are proteins and/or polypeptides having at least one PCL incorporated therein and at least one PYL incorporated therein, wherein PCL and PYL are produced biosynthetically and incorporated into the protein and/or polypeptide. .

Also provided herein are proteins and/or polypeptides having one or more PCL residues, wherein PCL is produced biosynthetically and incorporated into the protein and/or polypeptide, and wherein the one or more PCL residues is a label, dye, polymer, Water-soluble polymers, polyalkylene glycols, poly(ethylene glycol), derivatives of poly(ethylene glycol), sugars, lipids, photocrosslinkers, cytotoxic compounds, drugs, affinity labels, photoaffinity labels, reactive compounds; Resin, peptide, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelating agent, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, PCR probe, antisense polynucleotide, ribo-oligonucleotide, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labeling , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocausing moieties, actinic radiation excitable moieties, ligands, photoisomerizing moieties, biotin, biotin analogs , Heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-activators, amino thio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups , Chromophore group, chemiluminescent group, fluorescent moiety, electron high density group, magnetic group, intercalating group, chelating group, chromophore, energy transfer agent, biologically active agent, detectable label, small molecule, inhibitory ribonucleic acid, siRNA, radionucleotide, Neutron-capturing agent, biotin derivative, quantum dot(s), nanotransmitter, radiotransmitter, abzyme, enzyme, activated complex activator, virus, toxin, adjuvant, TLR2 agonist, TLR4 agonist, TLR7 efficacy Agent, TLR9 agonist, TLR8 agonist, T-cell epitope, phospholipid, LPS-like molecule, keyhole limpet hemocyanin (KLH), immunogenic hapten, aglycan, allergen, angiostatin, antihormonal, antioxidant, pressure Tamers, guide RNAs, saponins, shuttle vectors, macromolecules, mimotopes, receptors, reverse micelles, detergents, immune response enhancers, fluorescent dyes, FRET reagents, radio-imaging probes, other spectroscopic probes, prodrugs, toxins for immunotherapy , Solid support, -CH ₂ CH ₂ -(OCH ₂ CH ₂ O) _p -OX ² , and Proteins, and a group selected from _{- -O- (CH 2 CH 2 O} ) p CH 2 CH 2 -X 2 (8 alkyl, protecting group or a terminal functional group being wherein p is 1 to 10,000, X ² is H, C ₁₎ /Or is thereby derivatized by coupling to the polypeptide.

In certain embodiments of such proteins and/or polypeptides having one or more PYL residues incorporated therein, wherein PYL is produced biosynthetically and incorporated into the protein and/or polypeptide, pyrrolysine is one incorporated therein. For proteins and/or polypeptides having more than two PCL residues, they are derivatized thereby by coupling to the protein and/or polypeptide one of the aforementioned groups given above.

In certain embodiments of such proteins and/or polypeptides having at least one PCL and at least one PYL residue incorporated therein, wherein PCL and PYL are produced biosynthetically and incorporated into the protein and/or polypeptide, PCL And pyrrolysine is derivatized thereby by coupling to the protein and/or polypeptide one of the aforementioned groups given above for proteins and/or polypeptides having one or more PCL residues incorporated therein.

In certain embodiments, such above-mentioned biosynthesis occurs in eukaryotic cells, mammalian cells, yeast cells or insect cells. In certain embodiments, the cells are Escherichia coli cells, while in other embodiments, the yeast cells are Saccharomyces cerevisiae or Pichia pastoralis cells. In certain embodiments, the cells are CHO cells, HeLa cells, HEK293F cells or sf9 cells.

One aspect provided herein is a compound having a structure of formula I or II.

<Formula I>

<Formula II>

In the above formula,

R ₁ is H or an amino terminal modified group;

R ₂ is OH or a carboxy terminal modified group;

n is an integer from 1 to 5000;

Each AA is independently selected from amino acid residues, pyrrolysine analogue amino acid residues having the structure of formula A-2, and pyrrolysine analogue amino acid residues having the structure of formula B-2;

Each BB is an amino acid residue, a pyrrolysine analog having the structure of formula A-2, an amino acid residue of a pyrrolysine analog having the structure of formula B-2, an amino acid residue of a pyrrolysine analog having the structure of formula B-2, and a pyrrolysine analog having the structure of formula C-1 Amino acid residue, pyrrolysine analog amino acid residue having the structure of formula D-1, pyrrolysine analog amino acid residue having the structure of formula E-1, and pyrrolysine analog amino acid residue having the structure of formula F-1 , A pyrrolysine analog amino acid residue having a structure of the following formula G-1, a pyrrolysine analog amino acid residue having a structure of the following formula H-1, a pyrrolysine analog amino acid residue having a structure of the following formula I-1, Independently from the pyrrolysine analog amino acid residue having the structure of formula J-1, the pyrrolysine analog amino acid residue having the structure of formula K-1, and the pyrrolysine analog amino acid residue having the structure of formula L-1 Is selected;

In the above formula,

R _3, R ₅ and each R ₄ is H, -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX ¹ are independently selected;

R ₆ is H or C ₁ alkyl;

A is C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ heterocycloalkyl, 5-6 membered monocyclic aryl, 5-6 membered monocyclic heteroaryl, fused 9-10 membered bicyclic ring or fused 13 a to 14-membered tricyclic ring, where A is -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, Optionally substituted with 1 to 5 substituents independently selected from aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX ^1;

L is a bond, C _{1 - 8} alkylene, halo-substituted -C _{1 - 8} alkylene, hydroxy-substituted -C _{1 - 8} alkylene, C _2-8 alkenylene group, a halo-substituted -C _{2 - 8} alkenylene, hydroxy-substituted ₂ -C ₈ ^{alkenylene, -O (CR 11 R 12)} k -, -S (CR 11 R 12) k -, -S (O) k (CR 11 R ¹² ) _k -, -O(CR ¹¹ R ¹² ) _k -NR ¹¹ C(O)-, -O(CR ¹¹ R ¹² ) _k C(O)NR ¹¹ -, -C(O)-, -C( O)(CR ¹¹ R ¹² ) _k -, -C(S)-, -C(S)(CR ¹¹ R ¹² ) _k -, -C(O)NR ¹¹ -, -NR ¹¹ C(O)-, -NR ¹¹ (CR ¹¹ R ¹² ) _k -, -CONR ¹¹ (CR ¹¹ R ¹² ) _k -, -N(R ¹¹ )CO(CR ¹¹ R ¹² ) _k -, -C(O)NR ¹¹ (CR ^{11 _{R 12) k -, -NR 11}} C (O) (CR 11 R 12) k - is selected from wherein each R ¹¹ and R ¹² are independently H, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl or hydroxy-substituted -C _{1 - 8} is alkyl, k is an integer from 1 to 12;

X ¹ is a label, dye, polymer, water-soluble polymer, polyalkylene glycol, poly(ethylene glycol), derivative of poly(ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Label, reactive compound; Resin, peptide, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelating agent, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, PCR probe, antisense polynucleotide, ribo-oligonucleotide, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labeling , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocausing moieties, actinic radiation excitable moieties, ligands, photoisomerizing moieties, biotin, biotin analogs , Heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-activators, amino thio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups , Chromophore group, chemiluminescent group, fluorescent moiety, electron high density group, magnetic group, intercalating group, chelating group, chromophore, energy transfer agent, biologically active agent, detectable label, small molecule, inhibitory ribonucleic acid, siRNA, radionucleotide , Neutron-capturing agent, biotin derivative, quantum dot(s), nanotransmitter, radiotransmitter, abzyme, enzyme, activated complex activator, virus, toxin, adjuvant, TLR2 agonist, TLR4 agonist, TLR7 Agonist, TLR9 agonist, TLR8 agonist, T-cell epitope, phospholipid, LPS-like molecule, keyhole limpet hemocyanin (KLH), immunogenic hapten, aglycan, allergen, angiostatin, antihormonal, antioxidant, Aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, mimotopes, receptors, reverse micelles, detergents, immune response enhancers, fluorescent dyes, FRET reagents, radiation-imaging probes, other spectroscopic probes, prodrugs, for immunotherapy Toxin, solid support, -CH ₂ CH ₂ -(OCH ₂ CH ₂ O) _p -OX ² ,- _{O- (CH 2 CH 2 O)} p CH 2 CH 2 -X 2 , and are selected from any combination thereof, wherein p is 1 to 10,000, X ² is H, C _{1 - 8} alkyl, protecting group or terminal Is a functional group;

At least one AA is a pyrrolysine analog amino acid residue having the structure of Formula A-2 or Formula B-2, or at least one BB is Formula C-1 or Formula D-1 or Formula E-1 or Formula F- 1 or a pyrrolysine analog amino acid residue having the structure of formula G-1 or formula H-1 or formula I-1 or formula J-1 or formula K-1 or formula L-1.

In certain embodiments of the above compounds, Ring A is furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, pyrazole Zoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, dithian, thiomorpholine, pyridazine, pyrimidine , Pyrazine, piperazine, triazine, tritian, indolizine, indole, isoindole, indoline, benzofuran, benzothiophene, indazole, benzimidazole, benzthiazole, purine, quinolizine, quinoline, iso Quinoline, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine, quinuclidine, carbazole, acridine, phenazine, phenthiazin, phenoxazine, phenyl, indene, naphthalene, azulene , Fluorene, anthracene, phenanthracene, norborane and adamantine.

In other embodiments of the above compounds, Ring A is phenyl, furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole , Pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, ditian, thiomorpholine, pyridazine, It is selected from pyrimidine, pyrazine, piperazine, triazine and tritian.

In another embodiment of the above compound, Ring A is indolizine, indole, isoindole, indoline, benzofuran, benzothiophene, indazole, benzimidazole, benzthiazole, purine, quinolizine, quinoline, iso Quinoline, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine, quinuclidine, carbazole, acridine, phenazine, phenthiazine, phenoxazine, indene, naphthalene, azulene, flu Orene, anthracene, phenanthracene, norborane and adamantine.

In certain embodiments of these compounds, Ring A is selected from phenyl, naphthalene and pyridine.

In certain embodiments of the above compounds, each BB is an amino acid residue, a pyrrolysine analog amino acid residue having the structure of formula A-2, a pyrrolysine analog amino acid residue having a structure of formula B-2, formula C A pyrrolysine analog amino acid residue having a structure of -1, a pyrrolysine analog amino acid residue having a structure of the following formula D-2, a pyrrolysine analog amino acid residue having a structure of the following formula E-1, the following formula F-2 A pyrrolysine analog amino acid residue having a structure of, a pyrrolysine analog amino acid residue having a structure of the following formula G-1, a pyrrolysine analog amino acid residue having a structure of the following formula H-2, the structure of the following formula I-1 Having a pyrrolysine analog amino acid residue, a pyrrolysine analog amino acid residue having the structure of the following formula J-2, a pyrrolysine analog amino acid residue having the structure of the following formula K-1, and having the structure of the following formula L-2 It is independently selected from pyrrolysine analogue amino acid residues.

In the above formula,

R _3, R ₅ and each R ₄ is H, -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX ¹ are independently selected;

R ₆ is H or C ₁ alkyl;

When present, each R ₇ is -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, aryl, heteroaryl , Heterocycloalkyl or cycloalkyl, and -LX ¹ are independently selected;

L is a bond, C _{1 - 8} alkylene, halo-substituted -C _{1 - 8} alkylene, hydroxy-substituted -C _{1 - 8} alkylene, C _2-8 alkenylene group, a halo-substituted -C _{2 - 8} alkenylene, hydroxy-substituted ₂ -C ₈ alkenylene, polyalkylene glycols, poly (ethylene ^{glycol), -O (CR 11 R 12} ) k -, -S (CR 11 R 12) k - , -S(O) _k (CR ¹¹ R ¹² ) _k -, -O(CR ¹¹ R ¹² ) _k -NR ¹¹ C(O)-, -O(CR ¹¹ R ¹² ) _k C(O)NR ^11- , -C(O)-, -C(O)(CR ¹¹ R ¹² ) _k -, -C(S)-, -C(S)(CR ¹¹ R ¹² ) _k -, -C(O)NR ¹¹ -, -NR ¹¹ C(O)-, -NR ¹¹ (CR ¹¹ R ¹² ) _k -, -CONR ¹¹ (CR ¹¹ R ¹² ) _k -, -N(R ¹¹ )CO(CR ¹¹ R ¹² ) _k- , -C(O)NR ¹¹ (CR ¹¹ R ¹² ) _k -, -NR ¹¹ C(O)(CR ¹¹ R ¹² ) _k -, wherein each R ¹¹ and R ¹² is independently H, C _{1 - 8} alkyl, halo-substituted -C _1-8 alkyl, or hydroxy-substituted -C _{1 - 8} is alkyl, k is an integer from 1 to 12;

X ¹ is a label, dye, polymer, water-soluble polymer, polyalkylene glycol, poly(ethylene glycol), derivative of poly(ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Label, reactive compound; Resin, peptide, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelating agent, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, PCR probe, antisense polynucleotide, ribo-oligonucleotide, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labeling , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocausing moieties, actinic radiation excitable moieties, ligands, photoisomerizing moieties, biotin, biotin analogs , Heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-activators, amino thio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups , Chromophore group, chemiluminescent group, fluorescent moiety, electron high density group, magnetic group, intercalating group, chelating group, chromophore, energy transfer agent, biologically active agent, detectable label, small molecule, inhibitory ribonucleic acid, siRNA, radionucleotide , Neutron-capturing agent, biotin derivative, quantum dot(s), nanotransmitter, radiotransmitter, abzyme, enzyme, activated complex activator, virus, toxin, adjuvant, TLR2 agonist, TLR4 agonist, TLR7 Agonist, TLR9 agonist, TLR8 agonist, T-cell epitope, phospholipid, LPS-like molecule, keyhole limpet hemocyanin (KLH), immunogenic hapten, aglycan, allergen, angiostatin, antihormonal, antioxidant, Aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, mimotopes, receptors, reverse micelles, detergents, immune response enhancers, fluorescent dyes, FRET reagents, radiation-imaging probes, other spectroscopic probes, prodrugs, for immunotherapy Toxin, solid support, -CH ₂ CH ₂ -(OCH ₂ CH ₂ O) _p -OX ² ,- _{O- (CH 2 CH 2 O)} p CH 2 CH 2 -X 2 , and are selected from any combination thereof, wherein p is 1 to 10,000, X ² is H, C _{1 - 8} alkyl, protecting group or terminal It is a functional group.

In certain embodiments of these compounds, R ₆ is H, while in other embodiments of these compounds, R ₆ is C ₁ alkyl.

In certain embodiments of the above compounds, R ₅ is -LX ¹ . In certain embodiments of the above compounds, R ₇ is -LX ¹ . In certain embodiments of the above compounds, X ¹ is a sugar, polyethylene glycol, fluorescent moiety, immunomodulator, ribonucleic acid, deoxyribonucleic acid, protein, peptide, biotin, phospholipid, TLR7 agonist, immunogenic hapten or solid support. In certain embodiments of the above compounds, L is a poly (alkylene glycol), poly (ethylene glycol), C _{1 - 8} alkylene, halo-substituted -C _{1 - 8} alkylene or hydroxy-substituted -C _{1 8} is an alkylene group.

Another aspect provided herein is a method of derivatizing a protein comprising contacting a protein having a structure according to formula (I) with a reagent of formula (III) or (IV).

<Formula I>

In the above formula,

R ₁ is H or an amino terminal modified group;

R ₂ is OH or a carboxy terminal modified group;

n is an integer from 1 to 5000;

Each AA is independently selected from an amino acid residue, a pyrrolysine amino acid residue, a pyrrolysine analog amino acid residue having the structure of formula A-1, and a pyrrolysine analog amino acid residue having the structure of formula B-1, ;

R ₆ is H or C ₁ alkyl;

At least one AA is a pyrrolysine amino acid residue, or a pyrrolysine analog amino acid residue having the structure of Formula A-1 or Formula B-1.

<Formula III>

<Formula IV>

In the above formula,

R _3, R ₅ and each R ₄ is H, -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX ¹ are independently selected;

A is C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ heterocycloalkyl, 5-6 membered monocyclic aryl, 5-6 membered monocyclic heteroaryl, fused 9-10 membered bicyclic ring or fused 13 a to 14-membered tricyclic ring, where A is -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, Optionally substituted with 1 to 5 substituents independently selected from aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX ^1;

L is a bond, C _{1 - 8} alkylene, halo-substituted -C _{1 - 8} alkylene, hydroxy-substituted -C _{1 - 8} alkylene, C _2-8 alkenylene group, a halo-substituted -C _{2 - 8} alkenylene, hydroxy-substituted ₂ -C ₈ alkenylene group, a poly (alkylene glycol), poly (ethylene ^{glycol), -O (CR 11 R 12} ) k -, -S (CR 11 R 12) _k -, -S(O) _k (CR ¹¹ R ¹² ) _k -, -O(CR ¹¹ R ¹² ) _k -NR ¹¹ C(O)-, -O(CR ¹¹ R ¹² ) _k C(O)NR ¹¹ -, -C(O)-, -C(O)(CR ¹¹ R ¹² ) _k -, -C(S)-, -C(S)(CR ¹¹ R ¹² ) _k -, -C(O) NR ¹¹ -, -NR ¹¹ C(O)-, -NR ¹¹ (CR ¹¹ R ¹² ) _k -, -CONR ¹¹ (CR ¹¹ R ¹² ) _k -, -N(R ¹¹ )CO(CR ¹¹ R ¹² ) _k -, -C(O)NR ¹¹ (CR ¹¹ R ¹² ) _k -, -NR ¹¹ C(O)(CR ¹¹ R ¹² ) _k -, wherein each of R ¹¹ and R ¹² is independently H , C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl or hydroxy-substituted -C _{1 - 8} is alkyl, k is an integer from 1 to 12;

X ¹ is a label, dye, polymer, water-soluble polymer, polyalkylene glycol, poly(ethylene glycol), derivative of poly(ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Label, reactive compound; Resin, peptide, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelating agent, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, PCR probe, antisense polynucleotide, ribo-oligonucleotide, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labeling , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocausing moieties, actinic radiation excitable moieties, ligands, photoisomerizing moieties, biotin, biotin analogs , Heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-activators, amino thio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups , Chromophore group, chemiluminescent group, fluorescent moiety, electron high density group, magnetic group, intercalating group, chelating group, chromophore, energy transfer agent, biologically active agent, detectable label, small molecule, inhibitory ribonucleic acid, siRNA, radionucleotide , Neutron-capturing agent, biotin derivative, quantum dot(s), nanotransmitter, radiotransmitter, abzyme, enzyme, activated complex activator, virus, toxin, adjuvant, TLR2 agonist, TLR4 agonist, TLR7 Agonist, TLR9 agonist, TLR8 agonist, T-cell epitope, phospholipid, LPS-like molecule, keyhole limpet hemocyanin (KLH), immunogenic hapten, aglycan, allergen, angiostatin, antihormonal, antioxidant, Aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, mimotopes, receptors, reverse micelles, detergents, immune response enhancers, fluorescent dyes, FRET reagents, radiation-imaging probes, other spectroscopic probes, prodrugs, for immunotherapy Toxin, solid support, -CH ₂ CH ₂ -(OCH ₂ CH ₂ O) _p -OX ² ,- _{O- (CH 2 CH 2 O)} p CH 2 CH 2 -X 2 , and are selected from any combination thereof, wherein p is 1 to 10,000, X ² is H, C _{1 - 8} alkyl, protecting group or terminal It is a functional group.

In certain embodiments of the aforementioned methods, the amino acid residues of formula A-1 are amino acid residues having the structure of formula A-2 or formula A-3 below.

In certain embodiments of the aforementioned methods, the amino acid residues of formula B-1 are amino acid residues having the structure of formula B-2 or formula B-3 below.

In certain embodiments of the above-mentioned methods, the amino acid residues of formula A-1 are those of the amino acids of formula V, and the amino acid residues of formula B-1 are the residues of the amino acid formula VI:

(Wherein, R ₆ is H or C ₁ alkyl)

In certain embodiments, an amino acid of Formula V or Formula VI is produced biosynthetically within a cell comprising a pylB gene, a pylC gene, and a pylD gene, and the cell is in contact with a growth medium comprising a precursor. In other embodiments, the amino acids of Formula V or Formula VI are produced biosynthetically in cells comprising the pylC gene and the pylD gene, and the cell is in contact with a growth medium comprising the precursor.

In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula VII, and the precursor is ornithine, arginine, D-ornithine, D-arginine, (2S)-2-amino-6-(2,5 -Diaminopentanamido)hexanoic acid or (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid.

In certain embodiments, the amino acid of Formula VI is an amino acid having the structure of Formula VIII, the precursor is ornithine or arginine, and the precursor is ornithine, arginine, D-ornithine, D-arginine, (2S)-2- Amino-6-(2,5-diaminopentanamido)hexanoic acid or (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid.

In certain embodiments, the amino acid of formula V is an amino acid having the structure of formula VII and the precursor is D-ornithine or D-arginine. In certain embodiments, the amino acid of Formula VI is an amino acid having the structure of Formula VIII and the precursor is D-ornithine or D-arginine. In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula VII and the precursor is (2S)-2-amino-6-(2,5-diaminopentanamido)hexanoic acid. In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula VII and the precursor is (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid. In certain embodiments, the amino acid of Formula VI is an amino acid having the structure of Formula VIII and the precursor is (2S)-2-amino-6-(2,5-diaminopentanamido)hexanoic acid. In certain embodiments, the amino acid of Formula VI is an amino acid having the structure of Formula VIII, and the precursor is (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid.

In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula IX and the precursor is ornithine, arginine, D-ornithine, D-arginine, or 2,5-diamino-3-methylpentanoic acid.

In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula X and the precursor is ornithine, arginine, D-ornithine, D-arginine, or 2,5-diamino-3-methylpentanoic acid.

In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula IX and the precursor is D-2,5-diamino-3-methylpentanoic acid. In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula X and the precursor is D-2,5-diamino-3-methylpentanoic acid.

In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula IX and the precursor is (2R,3S)-2,5-diamino-3-methylpentanoic acid. In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula X and the precursor is (2R,3S)-2,5-diamino-3-methylpentanoic acid.

In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula IX and the precursor is (2R,3R)-2,5-diamino-3-methylpentanoic acid. In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula X and the precursor is (2R,3R)-2,5-diamino-3-methylpentanoic acid. In certain embodiments, the amino acid of formula V is an amino acid having the structure of formula IX, and the precursor is D-ornithine or D-arginine or (2S)-2-amino-6-((R)-2,5-di Aminopentanamido)hexanoic acid. In certain embodiments, the amino acid of formula V is an amino acid having the structure of formula X, and the precursor is D-ornithine or D-arginine or (2S)-2-amino-6-((R)-2,5-di Aminopentanamido)hexanoic acid.

In certain embodiments of the aforementioned methods, the amino acids of Formula V, Formula VI, Formula VII, Formula VII, Formula IX or Formula X are directed to orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA synthetase (O-RS). Is incorporated into an intracellular protein, wherein O-RS aminoacylates the O-tRNA to an amino acid of Formula V or Formula VI, and the O-tRNA recognizes one or more selector codons of the mRNA in the cell.

In certain embodiments of the above-mentioned method, the cell further comprises a pylS gene and a pylT gene, and the amino acid of Formula V, Formula VI, Formula VII, Formula VII, Formula IX or Formula X is an aminoacyl tRNA synthetase, and In the cell, it is incorporated into an intracellular protein by a tRNA that recognizes one or more selector codons of the mRNA, wherein the aminoacyl tRNA synthetase is a gene product of the pylS gene and the tRNA is a gene product of the pylT gene.

In certain embodiments of the aforementioned method, the selector codon is an amber codon (TAG).

In certain embodiments of the aforementioned methods, the cells are prokaryotic cells, while in other embodiments the cells are eukaryotic cells. In certain embodiments, the cells are Escherichia coli cells, while in other embodiments the cells are mammalian cells, yeast cells or insect cells. In certain embodiments, the yeast cells are Saccharomyces cerevisiae or Pichia pastoralis cells. In certain embodiments, the mammalian cells are CHO cells, HeLa cells, or HEK293F cells. In certain embodiments, the insect cells are sf9 cells.

Another aspect provided herein is a derivatized protein obtained using the above-mentioned method, wherein this derivatized protein has a structure according to formula II below.

<Formula II>

In the above formula,

R ₁ is H or an amino terminal modified group;

R ₂ is OH or a carboxy terminal modified group;

n is an integer from 1 to 5000;

Each BB is an amino acid residue, a pyrrolysine analog amino acid residue having the structure of the following formula A-1, a pyrrolysine analog amino acid residue having the structure of the formula B-1, A new analog amino acid residue, a pyrrolysine analog amino acid residue having a structure of the following formula D-1, a pyrrolysine analog amino acid residue having a structure of the following formula E-1, a pyrrolysine analog having a structure of the following formula F-1 Amino acid residue, a pyrrolysine analog amino acid residue having the structure of the following formula G-1, a pyrrolysine analog amino acid residue having the structure of the following formula H-1, a pyrrolysine analog amino acid residue having the structure of the following formula I-1 , Independent from a pyrrolysine analog amino acid residue having a structure of the following formula (J-1), a pyrrolysine analog amino acid residue having a structure of the following formula (K-1), and a pyrrolysine analog amino acid residue having a structure of formula L-1 Is selected as;

At least one BB is a formula C-1 or a formula D-1 or a formula E-1 or a formula F-1 or a formula G-1 or a formula H-1 or a formula I-1 or a formula J-1 or a formula K-1 or It is a pyrrolysine analog amino acid residue having the structure of formula L-1.

In the above-mentioned method and certain embodiments of said derivatized protein, Ring A is furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imida Zolidine, pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, ditian, thiomor Foline, pyridazine, pyrimidine, pyrazine, piperazine, triazine, tritian, indolizine, indole, isoindole, indoline, benzofuran, benzothiophene, indazole, benzimidazole, benzthiazole, purine, Quinolizine, quinoline, isoquinoline, cinoline, phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine, quinuclidine, carbazole, acridine, phenazine, phenthiazin, phenoxazine, phenyl , Indene, naphthalene, azulene, fluorene, anthracene, phenanthracene, norborane and adamantine.

In certain embodiments of the aforementioned methods and of the derivatized proteins, Ring A is phenyl, furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, Imidazolidine, pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, ditian, Selected from thiomorpholine, pyridazine, pyrimidine, pyrazine, piperazine, triazine and tritian.

In certain embodiments of the aforementioned methods and of the derivatized proteins, Ring A is indolizine, indole, isoindole, indoline, benzofuran, benzothiophene, indazole, benzimidazole, benzthiazole, purine, quie Nolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine, quinuclidine, carbazole, acridine, phenazine, pentazine, phenoxazine, indene, Naphthalene, azulene, fluorene, anthracene, phenanthracene, norborane and adamantine.

In the aforementioned method and certain embodiments of the derivatized protein, Ring A is selected from phenyl, naphthalyl and pyridyl.

In certain embodiments of the above-mentioned method, each BB is an amino acid residue, a pyrrolysine analog amino acid residue having the structure of formula A-2, a pyrrolysine analog amino acid residue having the structure of formula B-2, A pyrrolysine analog amino acid residue having a structure of C-1, a pyrrolysine analog amino acid residue having a structure of formula D-2, a pyrrolysine analog amino acid residue having a structure of formula E-1, the following formula F- A pyrrolysine analog amino acid residue having a structure of 2, a pyrrolysine analog amino acid residue having a structure of the following formula G-1, a pyrrolysine analog amino acid residue having a structure of the following formula H-2, A pyrrolysine analog amino acid residue having a structure, a pyrrolysine analog amino acid residue having a structure of the following formula J-2, a pyrrolysine analog amino acid residue having a structure of the following formula K-1, and the structure of the following formula L-2 It is independently selected from amino acid residues having pyrrolysine analogs.

In the above formula,

R _3, R ₅ and each R ₄ is H, -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX ¹ are independently selected;

R ₆ is H or C ₁ alkyl;

When present, each R ₇ is -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, aryl, heteroaryl , Heterocycloalkyl or cycloalkyl, and -LX ¹ are independently selected;

L is a bond, C _{1 - 8} alkylene, halo-substituted -C _{1 - 8} alkylene, hydroxy-substituted -C _{1 - 8} alkylene, C _2-8 alkenylene group, a halo-substituted -C _{2 - 8} alkenylene, hydroxy-substituted ₂ -C ₈ alkenylene, polyalkylene glycols, poly (ethylene ^{glycol), -O (CR 11 R 12} ) k -, -S (CR 11 R 12) k - , -S(O) _k (CR ¹¹ R ¹² ) _k -, -O(CR ¹¹ R ¹² ) _k -NR ¹¹ C(O)-, -O(CR ¹¹ R ¹² ) _k C(O)NR ^11- , -C(O)-, -C(O)(CR ¹¹ R ¹² ) _k -, -C(S)-, -C(S)(CR ¹¹ R ¹² ) _k -, -C(O)NR ¹¹ -, -NR ¹¹ C(O)-, -NR ¹¹ (CR ¹¹ R ¹² ) _k -, -CONR ¹¹ (CR ¹¹ R ¹² ) _k -, -N(R ¹¹ )CO(CR ¹¹ R ¹² ) _k- , -C(O)NR ¹¹ (CR ¹¹ R ¹² ) _k -, -NR ¹¹ C(O)(CR ¹¹ R ¹² ) _k -, wherein each R ¹¹ and R ¹² is independently H, C ₁ and _8, and the alkyl, k is an integer from 1 to 12, _{- 8} alkyl, halo-substituted -C _{1 - 8} alkyl or hydroxy-substituted -C ₁

X ¹ is a label, dye, polymer, water-soluble polymer, polyalkylene glycol, poly(ethylene glycol), derivative of poly(ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Label, reactive compound; Resin, peptide, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelating agent, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, PCR probe, antisense polynucleotide, ribo-oligonucleotide, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labeling , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocausing moieties, actinic radiation excitable moieties, ligands, photoisomerizing moieties, biotin, biotin analogs , Heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-activators, amino thio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups , Chromophore group, chemiluminescent group, fluorescent moiety, electron high density group, magnetic group, intercalating group, chelating group, chromophore, energy transfer agent, biologically active agent, detectable label, small molecule, inhibitory ribonucleic acid, siRNA, radionucleotide , Neutron-capturing agent, biotin derivative, quantum dot(s), nanotransmitter, radiotransmitter, abzyme, enzyme, activated complex activator, virus, toxin, adjuvant, TLR2 agonist, TLR4 agonist, TLR7 Agonist, TLR9 agonist, TLR8 agonist, T-cell epitope, phospholipid, LPS-like molecule, keyhole limpet hemocyanin (KLH), immunogenic hapten, aglycan, allergen, angiostatin, antihormonal, antioxidant, Aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, mimotopes, receptors, reverse micelles, detergents, immune response enhancers, fluorescent dyes, FRET reagents, radiation-imaging probes, other spectroscopic probes, prodrugs, for immunotherapy Toxin, solid support, -CH ₂ CH ₂ -(OCH ₂ CH ₂ O) _p -OX ² ,- _{O- (CH 2 CH 2 O)} p CH 2 CH 2 -X 2 , and are selected from any combination thereof, wherein p is 1 to 10,000, X ² is H, C _{1 - 8} alkyl, protecting group or terminal It is a functional group.

In certain embodiments of the aforementioned methods and of such derivatized proteins, R ₆ is H, while in other embodiments R ₆ is C ₁ alkyl.

In the aforementioned method and certain embodiments of the derivatized protein, R ₅ is -LX ¹ . In the above-mentioned method and certain embodiments of the derivatized protein, X ¹ is a sugar, polyethylene glycol, fluorescent moiety, immunomodulatory agent, ribonucleic acid, deoxyribonucleic acid, protein, peptide, biotin, phospholipid, TLR7 agonist, immunity. It is an original hapten or a solid support. In certain embodiments, L is a poly (alkylene glycol), poly (ethylene glycol), C _{1 - 8} alkylene, halo-substituted -C _{1 - 8} alkylene or hydroxy-substituted -C _{1 - 8} alkyl, It's Len.

In certain embodiments of the aforementioned methods, the reagent of formula IV is

이고, 여기서 L 및 X¹은 상기 기재된 바와 같다.

Where L and X ¹ are as described above.

In certain embodiments of the above reagents, L is a bond and X ¹ is polyethylene glycol.

In certain embodiments, the reagent of formula IV is

이고, PADRE는

이고, BG1은

이고, BG2는

이고, 여기서 *는 포스포티오에이트 연결부를 표시한다.Wherein the compound having at least one polyethylene glycol (PEG) moiety has an average molecular weight in the range of 1000 Da to 50 kDa, n is 20 to 1200, and exPADRE is

And PADRE is

And BG1 is

And BG2 is

And, where * represents a phosphorothioate linkage.

In certain embodiments, the reagent of Formula IV has the structure:

를 갖는 화합물이고, 여기서 화합물은 1000 Da 내지 30 kDa 범위의 평균 분자량을 갖고, n은 20 내지 679이다.

Wherein the compound has an average molecular weight ranging from 1000 Da to 30 kDa, and n is from 20 to 679.

In another embodiment, the reagent of Formula IV has the structure:

를 갖는 화합물이고, 여기서 화합물은 1000 Da 내지 45 kDa 범위의 평균 분자량을 갖고, n은 20 내지 1018이다.

Wherein the compound has an average molecular weight ranging from 1000 Da to 45 kDa, and n is from 20 to 1018.

Another aspect provided herein is a compound having a structure of Formula VII or Formula VIII, wherein the compound of Formula VII or Formula VIII is biosynthetically produced in a cell comprising a pylC gene and a pylD gene, and the cell is a precursor It is in contact with the growth medium containing.

<Formula VII>

<Formula VIII>

In certain embodiments of the above compounds, the cell comprises a pylB gene, a pylC gene and a pylD gene.

In certain embodiments of the above compounds, the precursor is ornithine or arginine, while in other embodiments the precursor is D-ornithine or D-arginine or (2S)-2-amino-6-((R)-2,5 -Diaminopentanamido)hexanoic acid.

In certain embodiments of the above compounds, the compound of Formula VII or Formula VIII is incorporated into an intracellular protein by an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS), wherein O-RS is O -tRNA is aminoacylated with a compound of Formula V or Formula VI, and the O-tRNA recognizes one or more selector codons of the mRNA in the cell.

In certain embodiments of the above compounds, the cell further comprises a pylS gene and a pylT gene, and the compound of Formula V or Formula VI is by an aminoacyl tRNA synthetase, and a tRNA that recognizes one or more selector codons of the mRNA in the cell. It is incorporated into an intracellular protein, wherein the aminoacyl tRNA synthetase is the gene product of the pylS gene and the tRNA is the gene product of the pylT gene.

In certain embodiments the selector codon is an amber codon (TAG). In certain embodiments, the cell is a prokaryotic cell, while in other embodiments the cell is a eukaryotic cell. In certain embodiments, the cells are Escherichia coli cells, while in other embodiments the cells are mammalian cells, yeast cells or insect cells. In certain embodiments, the yeast cells are Saccharomyces cerevisiae or Pichia pastoralis cells. In certain embodiments, the mammalian cells are CHO cells, HeLa cells, or HEK293F cells. In certain embodiments, the insect cells are sf9 cells.

Another aspect provided herein is a compound having the structure of Formula VII or Formula VIII, wherein the compound of Formula VII or Formula VIII is biosynthesized by a first cell in contact with a growth medium comprising a precursor and a second cell. It is produced and secreted by the antagonist, wherein the first cell is a feeder cell containing the pylC gene and the pylD gene.

<Formula VII>

<Formula VIII>

In certain embodiments of the above compounds, the first cell comprises a pylB gene, a pylC gene and a pylD gene.

In certain embodiments of the above compounds, the precursor is ornithine or arginine. In other embodiments of the above compounds, the precursor is D-ornithine or D-arginine. In another embodiment of the above compound, the precursor is (2S)-2-amino-6-(2,5-diaminopentanamido)hexanoic acid. In another embodiment of the above compound, the precursor is (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid.

In certain embodiments of the above compounds, the compound of Formula VII or Formula VIII is incorporated into a second intracellular protein by an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS), wherein the O-RS Aminoacylates the O-tRNA with a compound of Formula VII or Formula VIII, and the O-tRNA recognizes one or more selector codons of the mRNA in the second cell.

In certain embodiments of the above compounds, the second cell comprises a pylS gene and a pylT gene, and the compound of Formula VII or Formula VIII is incorporated into the protein in the second cell.

In certain embodiments of the above compounds, the compound of Formula VII or Formula VIII is incorporated into a second intracellular protein by an aminoacyl tRNA synthetase and a tRNA that recognizes one or more selector codons of the mRNA in the cell, wherein the aminoacyl tRNA Synthetase is the gene product of the pylS gene and tRNA is the gene product of the pylT gene.

In certain embodiments of the above compounds, the selector codon is an amber codon (TAG).

In certain embodiments of the above compounds, the first cell or the second cell is a prokaryotic cell. In certain embodiments of the above compounds, the first and second cells are prokaryotic cells. In certain embodiments of the above compounds, the first cell or the second cell is a eukaryotic cell. In certain embodiments of the above compounds, the first and second cells are eukaryotic cells.

In certain embodiments, the prokaryotic cell is an Escherichia coli cell. In certain embodiments, the eukaryotic cell is a mammalian cell, a yeast cell, or an insect cell. In certain embodiments, the yeast cells are Saccharomyces cerevisiae or Pichia pastoralis cells. In certain embodiments, the mammalian cells are CHO cells, HeLa cells, or HEK293F cells. In certain embodiments, the insect cells are sf9 cells.

Another aspect provided herein is a compound having the structure of Formula IX, wherein the compound of Formula IX is biosynthetically produced in cells containing the pylC gene and the pylD gene, and the cell is 2,5-diamino- It is in contact with a growth medium containing 3-methylpentanoic acid or D-2,5-diamino-3-methylpentanoic acid.

<Formula IX>

In certain embodiments of the above compounds, 2,5-diamino-3-methylpentanoic acid is (2R,3S)-2,5-diamino-3-methylpentanoic acid.

In certain embodiments of the above compounds, 2,5-diamino-3-methylpentanoic acid is (2R,3R)-2,5-diamino-3-methylpentanoic acid.

In certain embodiments of the above compounds, the cell comprises a pylB gene, a pylC gene and a pylD gene, and the cell is ornithine, arginine, D-ornithine, D-arginine, (2S)-2-amino-6-(2 ,5-diaminopentanamido)hexanoic acid 2,5-diamino-3-methylpentanoic acid or D-2,5-diamino-3-methylpentanoic acid.

In certain embodiments of the above compounds, the compound of formula IX is incorporated into an intracellular protein by an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS), wherein O-RS is an O-tRNA. Aminoacylated with a compound of formula IX, and the O-tRNA recognizes one or more selector codons of the mRNA in the cell.

In certain embodiments of the above compounds, the cell further comprises a pylS gene and a pylT gene, and the compound of formula IX is an intracellular protein by an aminoacyl tRNA synthetase, and a tRNA that recognizes one or more selector codons of the mRNA in the cell. Wherein the aminoacyl tRNA synthetase is the gene product of the pylS gene and the tRNA is the gene product of the pylT gene.

In certain embodiments, the selector codon is an amber codon (TAG). In certain embodiments, the cell is a prokaryotic cell, while in other embodiments, the cell is a eukaryotic cell. In certain embodiments, the prokaryotic cell is an Escherichia coli cell. In certain embodiments, the eukaryotic cell is a mammalian cell, a yeast cell, or an insect cell. In certain embodiments, the yeast cells are Saccharomyces cerevisiae or Pichia pastoralis cells. In certain embodiments, the mammalian cells are CHO cells, HeLa cells, or HEK293F cells. In certain embodiments, the insect cells are sf9 cells.

Another aspect provided herein is a compound having the structure of formula IX, wherein the compound of formula IX is 2,5-diamino-3-methylpentanoic acid or D-2,5-diamino-3-methylphene It is biosynthetically produced and secreted by first cells in contact with the growth medium containing carbonic acid and the second cells, wherein the first cells are feeder cells containing the pylC gene and the pylD gene.

<Formula IX>

In certain embodiments of the above compounds, 2,5-diamino-3-methylpentanoic acid is (2R,3S)-2,5-diamino-3-methylpentanoic acid.

In certain embodiments of the above compounds, 2,5-diamino-3-methylpentanoic acid is (2R,3R)-2,5-diamino-3-methylpentanoic acid.

In certain embodiments of the above compounds, the cell comprises a pylB gene, a pylC gene and a pylD gene, and the cell is ornithine, arginine, D-ornithine, D-arginine, (2S)-2-amino-6-(2 ,5-diaminopentanamido)hexanoic acid 2,5-diamino-3-methylpentanoic acid or D-2,5-diamino-3-methylpentanoic acid.

In certain embodiments of the above compounds, the compound of formula IX is incorporated into a second intracellular protein by an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS), wherein O-RS is O- The tRNA is aminoacylated with the compound of formula IX, and the O-tRNA recognizes one or more selector codons of the mRNA in the second cell.

In certain embodiments of the above compounds, the second cell comprises a pylS gene and a pylT gene, and the compound of formula IX is incorporated into the protein in the second cell.

In certain embodiments of the above compounds, the compound of formula IX is incorporated into a second intracellular protein by an aminoacyl tRNA synthetase, and a tRNA that recognizes one or more selector codons of the mRNA in the cell, wherein the aminoacyl tRNA synthetase is The gene product of the pylS gene and tRNA is the gene product of the pylT gene.

In certain embodiments, the selector codon is an amber codon (TAG).

In certain embodiments, the first cell or the second cell is a prokaryotic cell. In certain embodiments, the first and second cells are prokaryotic cells. In certain embodiments, the first cell or the second cell is a eukaryotic cell. In certain embodiments, the first and second cells are eukaryotic cells. In certain embodiments, the prokaryotic cell is an Escherichia coli cell. In certain embodiments the eukaryotic cell is a mammalian cell, a yeast cell or an insect cell. In certain embodiments, the yeast cells are Saccharomyces cerevisiae or Pichia pastoralis cells. In certain embodiments, the mammalian cells are CHO cells, HeLa cells, or HEK293F cells. In certain embodiments, the insect cells are sf9 cells.

Another aspect provided herein is a compound of formula IV,

이고, PADRE는

이고, BG1은

이고, BG2는

이고, 여기서 *는 포스포티오에이트 연결부를 표시한다.Wherein the compound having at least one polyethylene glycol (PEG) moiety has an average molecular weight in the range of 1000 Da to 50 kDa, n is 20 to 1200, and exPADRE is

And PADRE is

And BG1 is

And BG2 is

And, where * represents a phosphorothioate linkage.

Fig. 1. Structure of pyrrolysine (PYL) and pyrrolysine analog pyrroline-carboxy-lysine (PCL: PCL-A or PCL-B).
Figure 2. Comparison of possible biosynthetic pathways for pyrrolysine (A) and PCL (B).
Fig. 3. Aminoacylation of pylT tRNA by pyrrolysine (A) and PCL (B).
Fig. 4A. Plasmids containing pylT, pylS, pylB, pylC and pylD for incorporation of biosynthetically induced PCL or pyrrolysine in mammalian cells, and a single site TAG mutant gene construct for the model protein hRBP4.
Fig. 4B. Single site TAG mutant gene constructs for hRBP4, mEPO, hEPO and mIgG1. Single sites are indicated by arrows.
Fig. 5. Escherichia coli ) Plasmids containing pylT, pylS, pylB, pylC and pylD for incorporation of biosynthetically induced PCL or pyrrolysine in cells.
Fig. 6A. Expression of hRBP4 in HEK293F cells. TAG mutant construct of hRBP4 (#1-9). SDS-PAGE followed by Western blot by anti-His antibody. Arrows at 26 kDa indicate full length hRBP4.
Figures 6B and 6C. SDS-PAGE (A) and mass spectrum (B) of purified hRBP4 Phe62PCL produced in HEK293F cells in the presence of D-ornithine.
Figure 7. Mass spectrometric analysis of the trypsin digest of hRBP4 Phe122PCL shows the incorporation of PCL into the target Phe122TAG site. Designated MS/MS spectrum of YWGVASF*LQK (F* = PCL) (SEQ ID NO: 17).
Figure 8. Mass spectrum spectrometric analysis of trypsin digest of hRBP4 Phe122PCL (TIC and EIC of 2+ ions of YWGVASF*LQK) (SEQ ID NO: 17) shows the incorporation of PCL into the target Phe122TAG site.
Figure 9. Mass spectrometric analysis of trypsin digest of hRBP4 Phe122PCL. The mass spectrum shows the 3+ and 2+ precursors of YWGVASF*LQK (SEQ ID NO: 17) indicating incorporation of PCL into the target Phe122TAG site.
Figure 10. Detection of PCL biosynthesized from D-ornithine in lysates from HEK293F cells.
Figure 11. Incorporation of N-ε-cyclopentyloxycarbonyl-L-lysine (CYC) into various hRBP4 TAG mutant proteins in HEK293F cells. 11A shows CYC incorporation at various sites in hRBP4, as detected by SDS-PAGE, and Western blotting with anti-His-tag and anti-RBP4 antibodies. 11B shows the SDS-PAGE result of purified hRBP4 Phe62CYC (mutant #2). 11C shows the mass spectrum of hRBP4 Phe62CYC.
Figure 12. Mass spectrometric verification of CYC incorporation into the TAG site of the hRBP4 mutant Phe62CYC.
Figure 13. PCL incorporation as a function of various precursors and direct incorporation of various pyrrolysine analogs (including CYC) into hRBP4 TAG mutant proteins by HEK293F cells (Figs. 13A and 13B), and of biosynthetic genes pylB, pylC and pylD PCL incorporation using various combinations (Figure 13C). 13A is a Western blot of a sample that is not purified by an anti-His-tag antibody, and FIG. 13B is an SDS-PAGE gel of a Ni-NTA purified protein.
Figure 14. Potential precursors for PCL biosynthesis (A) and various pyrrolysine analogs (B).
Figure 15. Evaluation of various precursor and gene combinations for incorporation of PCL into FASTE in HK100 cells.
Figure 16. Potential biosynthetic scheme for PCL (PCL-A) formation.
Fig. 17A. Site-specific incorporation of biosynthetically generated PCL into a single TAG coding site (4 sites) of the Fc domain of mouse IgG1.
Fig. 17B. Site-specific incorporation of biosynthetically generated PCL into a single TAG coding site (11 sites) of erythropoietin (EPO) as detected by SDS-PAGE.
Figure 18. Site-specific incorporation of biosynthetically generated PCL into a single TAG coding site (2 sites) of the thioesterase domain of human fatty acid synthetase (FAS-TE). Figure 18 shows the mass spectrum (B) for SDS-PAGE (A) and PCL incorporation.
Fig. 19. Site-specific incorporation of biosynthetically generated PCL into a single TAG coding site (1 site) of FKBP-12. Figure 19 shows the mass spectrum for SDS-PAGE (A) and PCL incorporation (B) and the determination of FKBP12-I90PCL (C).
Fig. 20. Site-specific incorporation of biosynthetically generated PCL into a single TAG coding site (20 sites) of fibroblast growth factor 21 (FGF21). SDS-PAGE shows PCL incorporation into FGF21 at multiple sites.
Figure 21. Figure 21A shows the SDS-PAGE analysis of PYL- or PCL-incorporation into mTNF-α where glutamine Gln21 (CAA) has been mutated with the TAG stop codon in the presence and absence of pylB. Figure 21B SDS-PAGE evaluating the purity of the protein. Figure 21C is a circular mass spectrum of mEGF Tyr10TAG expressed in Escherichia coli, indicating that PYL and PCL were incorporated into a mixture of proteins (Figure 21C, bottom), and PCL was predominantly incorporated (Figure 21C, top). .
Figure 22. Possible reaction scheme for chemical derivatization of PCL with 2-amino-benzaldehyde.
Figure 23. Assumed protein conjugate and mass change after derivatization of PCL with 2-amino benzaldehyde, 2-aminoacetophenone and 2-amino-5-nitro-benzophenone.
Figure 24. Mass spectrometric analysis of hRBP4 Phe122PCL derivatized with 2-amino-benzaldehyde (2-ABA).
Figure 25. Mass spectrometric analysis of the trypsin digest of the 2-ABA-derivatized hRBP4 Phe122PCL protein demonstrates derivatization of PCL residues incorporated in the TAG site. YWGVASF*LQK peptide (SEQ ID NO: 17).
Fig. 26. Evaluation of pH dependence of derivatization of hRBP4 Phe122PCL by 2-ABA.
Figure 27. Evaluation of reaction efficiency as a function of ratio of reactants to protein concentration, and reactivity with 2-ABA, 2-ANBP and 2-AAP.
Fig. 28. Derivatization for hRBP4 (B: 15400-fold excess) with OMePhe and OMePhe at a molar ratio exceeding 4700 times (A: 4700 times excess of 2-ABA compared to protein).
Figure 29. Derivatization of FAS-TE Tyr2454PCL with 2-amino-acetophenone (2-AAP). Mass spectra of unreacted samples (A and C) and samples derivatized with 2-AAP at pH 5.0 (B) and pH 7.4 (D).
Figure 30. General scheme for site-specific modification of proteins through chemical derivatization of pyrrolysine and/or PCL by 2-amino-benzaldehyde or 2-amino-benzaldehyde analogs.
Figure 31. Illustrative of an embodiment of a functionalized PEG polymer, 2-amino-acetophenone-PEG8 (2-AAP-PEG8; TU3205-044), coupled to a protein via a PCL residue incorporated into the protein.
Fig. 32. Derivatization of hRBP4 Phe122PCL by 2-AAP-PEG8. PCL incorporated at position 122 compared to the mass spectra of unreacted hRBP4 Phe122PCL protein (C), and wild-type hRBP4 (D and E) with 2-AAP-PEG8 added and wild-type hRBP4 without 2-AAP-PEG8 added. Mass spectra at pH 7.5 (A) and pH 5.0 (B) after derivatization of hRBP4 with 2-AAP-PEG8 having.
Figure 33. Derivatization of FAS-TE Tyr2454PCL by 2-AAP-PEG8. Mass spectra for unreacted protein (A), and FAS-TE Tyr2454PCL protein (B) reacted with 2-AAP-PEG8 (TU3205-044). Figures 33C and 33D show the derivatization of FAS-TE Tyr2454PCL by 2.4 kDa 2-AAP-PEG (TU3205-048) (Figure 33C at room temperature, Figure 33D at 4°C).
Fig. 34. PEGylation of FAS-TE Tyr2454PCL with 0.5 kDa 2-AAP-PEG (2-AAP-PEG8), 2.4 kDa 2-AAP-PEG and 23 kDa 2-AAP-PEG at the molar ratios shown.
Figure 35. Derivatization of FGF21 Lys81PCL by 2-AAP-PEG8. Mass spectrum of unreacted FGF21 Lys84PCL (A) and FGF21 Lys84PCL (B) after derivatization with 2-AAP-PEG8.
Figure 36. PEGylation of FGF21 protein. After derivatization of 7 FGF21 PCL mutants with 23 kDa 2-AAP-PEG showing PEG-FGF21, full length (FL) FGF21-PCL and truncated (TR) FGF21-PCL, before partial purification (A) and The SDS-PAGE result obtained after (B).
Figure 37. PEGylation of EPO protein. SDS-PAGE after derivatization of mouse EPO PCL mutants by 23 kDa 2-AAP-PEG.
Figure 38. Derivatization of PCL with amino sugars. Generalized reaction scheme in which a protein with PCL incorporated therein ( _{represented as R 1} ) is coupled with D-mannosamine.
Fig. 39. Derivatization of hRBP4 Phe122PCL by D-mannosamine. Mass spectrum of hRBP4 with PCL incorporated at position 122 after reaction with mannosamine.
Fig. 40. Derivatization of FAS-TE Leu2222PCL by D-mannosamine. Mass spectrum of unreacted human fatty acid synthetase (FAS-TE) (FAS-TE Leu2222PCL/Leu2223Ile) with PCL incorporated at position 2222 (A) and protein reacted with mannosamine (B).
Figure 41. Illustration of an embodiment for site specific attachment of an oligosaccharide to a protein via reacting a 2-ABA moiety linked to an oligosaccharide with PCL incorporated into the protein.
Figure 42. Illustration of certain embodiments of protein-protein conjugates (heterodimers, heterotrimers, homotrimers) formed by crosslinked proteins with PCL incorporated therein.
Figure 43. Homodimer formation by PCL-specific, bifunctional crosslinkers as exemplified for the FGF21 Lys84PCL protein. A non-limiting example of the bifunctional crosslinking agent used to form the homodimer is shown in A, and the mass spectrum of the reaction mixture of FGF-21 Lys84PCL crosslinked using the bifunctional linker is shown in B.
Fig. 44. Homodimer formation of FGF-21 PCL mutant protein by bifunctional crosslinking agent. Figure 44A shows the mass spectrum of crosslinked FGF-21, where the reaction conditions are changed from the conditions used in Figure 43.
Figure 45. Embodiments for crosslinking agents used to form trimers.
Figure 46. Illustration of various embodiments of site specific labeling and labeling using the methods provided herein.
Figure 47. Figure 47A shows the ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with biotin. Figure 47B shows a Western blot of mEGF-Y10PCL-ABA-biotin conjugate using horseradish peroxidase (HRP) conjugated goat anti-biotin antibody. Figure 47C shows the ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with fluorescein. Figure 47D shows the ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with disaccharide.
Figure 48. Figure 48A shows the ESI mass spectrometric analysis of mTNF-Q21PCL conjugated with mono-nitrophenyl hapten. 48B shows the ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with mono-nitrophenyl hapten. 48C shows the ESI mass spectrometric analysis of mTNF-Q21PCL conjugated with di-nitrophenyl hapten. 48D shows the ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with di-nitrophenyl hapten.
Figure 49. Figure 49A shows the ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with a TLR7 agonist, and Figure 49B shows the ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with a phospholipid.
Figure 50. Figure 50A and Figure 50B show MALDI-TOF mass spectrometric analysis of mTNF-Q21PCL conjugated with PX2-PADRE at two different pH values (Figure 50A: pH 5.0; Figure 50B: pH 7.5). Figure 50C shows the ESI mass spectrometric analysis of mTNF-Q21PCL conjugated with BHA-exPADRE.
Fig. 51. Fig. 51 is an ESI mass spectrum showing the coupling of BHA-exPADRE to mEGF-Y10PCL.
Figure 52. Figure 52A is a gel shift assay of the coupling of BHA-BG1 (7.4 kDa) and BHA-BG2 (7.4 kDa) to mTNF-Q21PCL (19.3 kDa). Figure 52B is a gel shift assay of the coupling of BHA-BG2 (7.4 kDa) to mEGF-Y10PCL (7.2 kDa).
Figure 53. Figure 53 illustrates an embodiment of this site specific orientation attachment.
Figure 54. Figure 54A shows the ESI mass spectrometric analysis of hFGF21-K150PCL coupled to 2-ABA and then _{reduced with 20 mM NaCNBH 3 for 1 hour.} 54B shows the ESI mass spectrometric analysis of the reduced hFGF21-K150PCL 2-ABA conjugate after dialysis with 10 mM phosphate buffer (pH 7.5) and incubation at 50° C. for 1 day.
Figure 55. Figure 55 demonstrates the stability of the PCL linkage to PEGylated FGF21 in the presence and absence of reduction _{with NaCNBH 3.} SDS-PAGE gels for the reduced and non-reduced samples are shown in Figure 55A. Additionally, Figure 55B shows SDS-PAGE gels for non-reduced samples incubated at 4°C, room temperature, 37°C and 50°C, and 95°C for 60 hours.
Figure 56. NMR analysis of PCL-A reaction with 2-ABA.
Figure 57. Figure 57A is the proposed structure of the product resulting from the reaction between PCL-A and 2-ABA. 57B shows the proposed equilibrium structure of the product resulting from the reaction between PCL-A and 2-ABA. 57C is the proposed structure of the reduced product.
Fig. 58. NMR analysis of PCL-B reaction with 2-ABA.
Figure 59. Derivatization of pyrrolysine (Pyl) and PCL incorporated in mEGF.

Provided herein are methods and compositions used to site-specifically modify proteins, polypeptides and/or peptides, wherein such methods are genetically encoded pyrrolysine or pyrroline-carboxy-lysine (PCL) residues, wherein , Pyrrolysine and PCL amino acids are produced biosynthetically). Various types of molecules are provided herein that are site-specifically coupled to proteins, polypeptides, and/or peptides having one or more PCL residues or pyrrolysine biosynthetically incorporated therein. In certain embodiments, such site-specific modifications are used to site-specifically label proteins, polypeptides and/or peptides. In certain embodiments, the label is a fluorescent moiety, a phosphorescent moiety, a chemiluminescent moiety, a chelating moiety, an intercalating moiety, a radioactive moiety, a chromophore moiety, a radioactive moiety, a rotational label moiety, an NMR-active moiety, a PET or MRI imaging reagent. In certain embodiments, such site-specific modifications are used to attach immune modulators to proteins, polypeptides and/or peptides. In other embodiments, such site specific modifications are used to attach poly(ethylene glycol) (PEG) to proteins, polypeptides and/or peptides. In other embodiments, such site specific modifications are used to attach (glycosylate) sugars to proteins, polypeptides and/or peptides.

In other embodiments, such site-specific modifications are used to crosslink proteins, polypeptides and/or peptides, thereby forming heterooligomers, including, but not limited to, heterodimers and heterotrimers. In certain embodiments, such site-specific modifications are used to site-specifically cross-link the antibody to proteins, polypeptides and/or peptides. In other embodiments, such site-specific modifications crosslink proteins, polypeptides and/or peptides, whereby protein-protein conjugates, protein-polypeptide conjugates, protein-peptide conjugates, polypeptide-polypeptide conjugates, polypeptide-peptide conjugates or peptides. -Used to form peptide conjugates.

In other embodiments, such site-specific modifications are used to site-specifically link the antibody to a toxic protein provided herein, thereby forming an antibody-drug conjugate. In other embodiments, such site-specific modifications are used to site-specifically link an antibody coupled to a low molecular weight drug to a protein, thereby forming an antibody-drug conjugate.

In other embodiments, such site specific modifications are used to site-specifically link a receptor-ligand to a toxic protein provided herein, thereby forming a receptor-ligand-drug conjugate. In other embodiments, such site specific modifications are used to site-specifically link a receptor-ligand coupled to a low molecular weight drug to a protein, thereby forming a receptor-ligand-drug conjugate.

Proteins, polypeptides and/or peptides having pyrrolysine and/or PCL incorporated therein using the methods provided are provided herein. These proteins include erythropoietin (EPO), fibroblast growth factor 21 (FGF21), interferon alpha (INF-α), interleukin 2 (IL-2), interleukin 4 (IL-4), and interleukin 6 (IL-6). , Interleukin 10 (IL-10), interleukin 17 (IL-17), insulin-like growth factor 1 (IGF-1) and interferon beta (INF-β).

Provided herein are proteins, polypeptides and/or peptides having pyrrolysine and/or PCL incorporated therein using the methods provided herein and further derivatized using the methods provided herein. Such derivatization includes, but is not limited to, PEGylation. These proteins include erythropoietin (EPO), fibroblast growth factor 21 (FGF21), interferon alpha (INF-α), interleukin 2 (IL-2), interleukin 4 (IL-4), and interleukin 6 (IL-6). , Interleukin 10 (IL-10), interleukin 17 (IL-17), insulin-like growth factor 1 (IGF-1) and interferon beta (INF-β).

Further provided herein are proteins, polypeptides and/or peptides crosslinked using the methods provided herein, having pyrrolysine and/or PCL incorporated therein. Such proteins include, but are not limited to, erythropoietin (EPO), fibroblast growth factor 21 (FGF21), interferon alpha (INF-α) and interferon beta (INF-β).

In other embodiments, such site-specific modification is a protein in which the position of the site-specifically incorporated pyrrolysine or PCL allows for controlled orientation and attachment of the protein, polypeptide and/or peptide onto the solid support surface. , Polypeptides and/or peptides. In certain embodiments, such solid supports are coated or uncoated plastic microtiter plates, glass slides, silica surfaces, polymer beads, gold particles or nano-particles. In certain embodiments, such controlled orientation and attachment are used for analysis of proteins, polypeptides and peptides by ELISA or other antibody assays. In certain embodiments, such controlled orientation and attachment are used to purify and/or identify ligands of immobilized proteins, polypeptides and/or peptides. In certain embodiments, such controlled orientation and attachment can be achieved by means of evanescent wave analysis, including but not limited to, assays that do not employ labeling using surface plasmon resonance analysis. And to analyze their interactions. In certain embodiments, such controlled orientation and adhesion on a surface is characterized by microbalance (electrical, optical and/or mechanical), infrared spectroscopy, Raman spectroscopy (surface enhanced Raman spectroscopy, vanishing resonance, fluorescence, interferometry). , Mass spectrometry and other spectroscopy) are used to analyze proteins, polypeptides and peptides, and their interactions. Such assay methods include immobilized proteins, polypeptides and/or peptides of other proteins, polypeptides, peptides, nucleic acids, DNA, RNA, small molecules, drugs, metabolites, sugars, carbohydrates, oligosaccharides, polysaccharides and/or other It is used to investigate interactions with molecules (including changes in conformation caused by these interactions). These assay methods also investigate the interaction of immobilized proteins, polypeptides and/or peptides with multiple sub-unit protein complexes, study natural, recombinant, synthetic or tagged recombinant molecules, and in body fluids, cell culture. Discover new interaction partners in supernatant or unprocessed extracts of water, study the interaction of small molecules, such as drug candidates, with their targets, and use natural membranes, artificial membranes or vesicles to interact with membrane biochemistry or membrane-boundary receptors. Study the action, investigate replication, transcription and translation, determine the molecular relationship during the formation of protein complexes and their interactions with DNA, study the hybridization of DNA and RNA, and interactions involving whole cells or viruses , Study the effect of glycosylation on molecular interactions, and determine the specific recognition properties of cell surface carbohydrates.

In other embodiments, such site-specific modifications are used to site-specifically attach nucleic acids to proteins. In certain embodiments, such site-specific modifications are used to site-specifically attach nucleic acids to antibodies or antibody fragments. In certain embodiments, the nucleic acid attached to the protein or antibody is used to immobilize the protein or antibody at defined locations on the DNA array through hybridization. In certain embodiments, the nucleic acid attached to the protein or antibody is PCR, strand displacement amplification (SDA), ligase chain reaction (LCR), contiguous ligation immunity-PCR, rolling circle amplification, transcription-mediated amplification, NEN's thyramide signal It is used to detect protein or antibody binding through amplification or other signal amplification methods. In certain embodiments, this site-specific attachment of a PCR probe to an antibody is used to generate reagents for an immuno-PCR reaction (M. Adler, R. Wacker, Ch.M. Niemeyer, Sensitivity by combination). : Immuno-PCR and related technologies, Analyst, 2008, 133, 702-718). In certain embodiments, the nucleic acid attached to the protein or antibody enables simultaneous immuno-PCR or other immuno-assay (multiplexed immuno-PCR or multiplexed immuno-assay) of multiple analytes.

In certain embodiments, the nucleic acid attached to the protein or antibody modulates the formation of homo- and heterodimers.

Justice

As used herein, the term “alkyl” refers to a saturated branched or straight chain hydrocarbon. As used herein, the terms "C ₁ -C ₃ alkyl", "C ₁ -C ₄ alkyl", "C ₁ -C ₅ alkyl", "C ₁ -C ₆ alkyl", "C ₁ -C ₇ alkyl" and “C ₁ -C ₈ alkyl” refers to an alkyl group containing at least 1 and up to 3, 4, 5, 6, 7 or 8 carbon atoms, respectively. Non-limiting examples of alkyl groups as used herein include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, hexyl, heptyl, octyl, Nonyl, decyl, etc. are included.

The term “alkylene” as used herein refers to a saturated branched or straight chain divalent hydrocarbon radical, wherein the radical is derived by removing one hydrogen atom from each of the two carbon atoms. As used herein, the terms "C ₁ -C ₃ alkylene", "C ₁ -C ₄ alkylene", "C ₁ -C ₅ alkylene" and "C ₁ -C ₆ alkylene" are at least one, and It refers to an alkylene group containing up to 3, 4, 5 or 6 carbon atoms, respectively. Non-limiting examples of alkylene groups used herein include methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, t-butylene, n-pentylene, isopentylene , Hexylene, and the like are included.

As used herein, the term “alkoxy” refers to the group _{—OR a , wherein R a} is an alkyl group as defined herein. As used herein, the terms "C ₁ -C ₃ alkoxy", "C ₁ -C ₄ alkoxy", "C ₁ -C ₅ alkoxy", "C ₁ -C ₆ alkoxy", "C ₁ -C ₇ alkoxy" and “C ₁ -C ₈ alkoxy” refers to an alkoxy group containing at least one alkyl moiety and up to 3, 4, 5, 6, 7 or 8 carbon atoms, respectively. Non-limiting examples of alkoxy groups as used herein include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, pentoxy, hexoxy, heptoxy, and the like.

As used herein, the term “amino terminal modifying group” refers to any molecule that forms a linkage with a terminal amine group. By way of example, such terminal amine groups include, but are not limited to, amine protecting groups, the ends of polymer molecules (wherein such polymer molecules include polypeptides, polynucleotides and polysaccharides, etc.). The amino terminal modification group also includes, but is not limited to, a variety of water soluble polymers, peptides or proteins. By way of example only, terminal modifying groups include polyethylene glycol or serum albumin. Certain amino terminal modifying groups are used to modify the therapeutic properties of proteins, including but not limited to increasing serum half-life.

As used herein, the term "aryl" has a total of 5 to 14 ring members, at least one ring in the system is aromatic, and each ring in the system contains 3 to 7 ring members, monocyclic, bicyclic And tricyclic ring systems. Aryl groups are “optionally substituted”, wherein such aryl groups contain one or more substituents. Unless otherwise defined herein, suitable substituents are generally halogen, -R, -OR, -SR, -NO ₂ , -CN, -N(R) ₂ , -NRC(O)R, -NRC(S) R, -NRC(O)N(R) ₂ , -NRC(S)N(R) ₂ , -NRCO ₂ R, -NRNRC(O)R, -NRNRC(O)N(R) ₂ , -NRNRCO ₂ R, -C(O)C(O)R, -C(O)CH ₂ C(O)R, -CO ₂ R, -C(O)R, -C(S)R, -C(O) N(R) ₂ , -C(S)N(R) ₂ , -OC(O)N(R) ₂ , -OC(O)R, -C(O)N(OR)R, -C(NOR )R, -S(O) ₂ R, -S(O) ₃ R, -SO ₂ N(R) ₂ , -S(O)R, -NRSO ₂ N(R) ₂ , -NRSO ₂ R,- N(OR)R, -C(=NH)-N(R) ₂ , -P(O) ₂ R, -PO(R) ₂ , -OPO(R) ₂ , -(CH ₂ ) _0-2 NHC (O)R, phenyl (Ph) optionally substituted with R, -O(Ph) optionally substituted with R, -(CH ₂ ) _1-2 (Ph) optionally substituted with R, or- CH=CH(Ph), wherein each independently appearing R is hydrogen, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ alkoxy, unsubstituted 5-6 membered heteroaryl, Two Rs selected from phenyl, -O(Ph) or -CH ₂ (Ph), or independently appearing on the same or different substituents, are combined with the atom(s) to which each R is attached, and nitrogen, oxygen , Or an optionally substituted 3-12 membered saturated, partially unsaturated, or fully unsaturated monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from sulfur. Non-limiting examples of aryl groups as used herein include phenyl, naphthyl, fluorenyl, indenyl, azulenyl, anthracenyl, and the like.

The term "arylene" as used refers to a divalent radical derived from an aryl group.

As used herein, “bifunctional linker” (also referred to as “bifunctional polymer”) refers to a linker comprising two functional groups capable of reacting specifically with other moieties to form a covalent or non-covalent bond. Such residues include, but are not limited to, amino acid side chain groups. By way of example only, the bifunctional linker has a functional group reactive with the group on the first peptide, and another functional group reactive with the group on the second peptide, and thus a conjugate comprising a first peptide, a bifunctional linker and a second peptide To form. The bifunctional linker has any desired length or molecular weight and is selected to provide the specific spacing or configuration desired.

As used herein, “multifunctional linker” (also referred to as “multifunctional polymer”) refers to a linker comprising two or more functional groups capable of reacting with other moieties to form covalent or non-covalent bonds. Such residues include, but are not limited to, amino acid side chain groups. The multifunctional linker has any desired length or molecular weight and is selected to provide the specific spacing or configuration desired.

As used herein, the term "cyano" refers to the group -CN.

The term “cycloalkyl,” as used herein, refers to a saturated or partially unsaturated, monocyclic, fused bicyclic, fused tricyclic or bridged polycyclic ring assembly. As used herein, the terms "C ₃ -C ₅ cycloalkyl", "C ₃ -C ₆ cycloalkyl", "C ₃ -C ₇ cycloalkyl", "C ₃ -C ₈ cycloalkyl", "C ₃ -C ₉ Cycloalkyl" and "C ₃ -C ₁₀ Cycloalkyl" means 3 or more saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assemblies, and 5, 6, 7, 8, It refers to a cycloalkyl group containing up to 9 or 10 carbon atoms. Non-limiting examples of cycloalkyl groups as used herein include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopentenyl, cyclohexenyl, decahydronaphthalenyl, 2,3,4,5,6,7-hexahydro-1H-indenyl, and the like.

As used herein, the term “cyclodextrin” refers to a cyclic carbohydrate consisting of 6 to 8 or more glucose molecules in ring formation. The outside of the ring contains water-soluble groups, and at the center of the ring is a relatively non-polar cavity capable of accommodating small molecules.

The term “halogen” as used herein refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

The term “halo” as used herein refers to halogen radicals (fluoro (-F), chloro (-Cl), bromo (-Br) and iodo (-I)).

As used herein, the term "haloacyl" contains halogen moieties including, but not limited to _{, -C(O)CH 3} , -C(O)CF ₃ , -C(O)CH ₂ OCH _{3, etc.} Refers to an acyl group.

The term “haloalkyl” or “halo-substituted alkyl” as used herein refers to an alkyl group as defined herein substituted with one or more halo groups or combinations thereof. As used herein, non-limiting examples of such branched or straight-chain haloalkyl groups include, but are not limited to, trifluoromethyl, pentafluoroethyl, and the like, substituted with one or more halo groups or combinations thereof. Methyl, ethyl, propyl, isopropyl, isobutyl and n-butyl.

The term “haloalkoxy,” as used herein, refers to an alkoxy group, as defined herein, substituted with one or more halo groups or combinations thereof. As used herein, non-limiting examples of such branched or straight-chain haloalkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, substituted with one or more groups or combinations thereof. t-butoxy, pentoxy, hexoxy, heptoxy, and the like.

The term “heteroalkyl,” as used herein, refers to an alkyl group as defined herein in which one or more carbon atoms have been independently replaced by one or more oxygen, sulfur, nitrogen, or combinations thereof.

The term “heteroalkylene” as used herein refers to a divalent radical derived from heteroalkyl.

The term “heteroaryl” as used herein has a total of 5 to 14 ring members, at least one ring in the system is aromatic, and at least one ring in the system contains at least one heteroatom selected from nitrogen, oxygen and sulfur. And refers to a monocyclic, bicyclic and tricyclic ring system in which each ring in the system contains 3 to 7 ring members. Unless defined otherwise above and herein, suitable substituents on the unsaturated carbon atom of the heteroaryl group are generally halogen, -R, -OR, -SR, -NO ₂ , -CN, -N(R) ₂ , -NRC (O)R, -NRC(S)R, -NRC(O)N(R) ₂ , -NRC(S)N(R) ₂ , -NRCO ₂ R, -NRNRC(O)R, -NRNRC(O )N(R) ₂ , -NRNRCO ₂ R, -C(O)C(O)R, -C(O)CH ₂ C(O)R, -CO ₂ R, -C(O)R, -C (S)R, -C(O)N(R) ₂ , -C(S)N(R) ₂ , -OC(O)N(R) ₂ , -OC(O)R, -C(O) N(OR)R, -C(NOR)R, -S(O) ₂ R, -S(O) ₃ R, -SO ₂ N(R) ₂ , -S(O)R, -NRSO ₂ N( R) ₂ , -NRSO ₂ R, -N(OR)R, -C(=NH)-N(R) ₂ , -P(O) ₂ R, -PO(R) ₂ , -OPO(R) ₂ , -(CH ₂ ) _0-2 NHC(O)R, phenyl (Ph) optionally substituted with R, -O(Ph) optionally substituted with R, -(CH ₂ ) _1-2 ( Ph), or -CH=CH(Ph) optionally substituted with R, wherein each independently appearing R is hydrogen, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ alkoxy, Unsubstituted 5 to 6 membered heteroaryl, phenyl, -O (Ph) or -CH ₂ (Ph), or two R independently appearing on the same or different substituents, each R is bonded Combined with the atom(s) to form an optionally substituted 3-12 membered saturated, partially unsaturated or fully unsaturated monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Non-limiting examples of heteroaryl groups as used herein include benzofuranyl, benzofurazanyl, benzoxazolyl, benzopyranyl, benzthiazolyl, benzothienyl, benzazepinyl, benzimidazolyl, benzothiopyra Neil, benzo[1,3]dioxole, benzo[b]furyl, benzo[b]thienyl, cinnolinyl, furazinyl, furyl, furopyridinyl, imidazolyl, indolyl, indozinyl, indoline 2-one, indazolyl, isoindolyl, isoquinolinyl, isoxazolyl, isothiazolyl, 1,8-naphthyridinyl, oxazolyl, oxaindolyl, oxadiazolyl, pyrazolyl, pyrrolyl, Phthalasinyl, pteridinil, purinyl, pyridyl, pyridazinyl, pyrazinyl, pyrimidyl, pyrimidinyl, quinoxalinyl, quinolinyl, quinazolinyl, 4H-quinolinyl, thiazolyl, thia Diazolyl, thienyl, triazinyl, triazolyl and tetrazolyl.

As used herein, the term "heterocycloalkyl" refers to one or more of the ring carbons being -O-, -N=, -NR-, -C(O)-, -S-, -S(O)-, or -S (O) _2- (wherein R is hydrogen, C ₁ -C ₄ alkyl or nitrogen protecting group), provided that the ring of the group does not contain two adjacent O or S atoms Refers to cycloalkyl as defined in. Non-limiting examples of heterocycloalkyl groups as used herein include morpholino, pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinylone, 1,4-dioxa-8- Aza-spiro[4.5]des-8-yl, 2H-pyrrolyl, 2-pyrrolinyl, 3-pyrrolinyl, 1,3-dioxolanyl, 2-imidazolinyl, imidazolidinyl, 2 -Pyrazolinyl, pyrazolidinyl, 1,4-dioxanyl, 1,4-ditianyl, thiomorpholinyl, azepanyl, hexahydro-1,4-diazepinyl, tetrahydrofuranyl, dihydro Furanyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, thioxanyl, azetidinyl, oxetanyl, thietanyl, oxepanyl, thiepanyl, 1,2, 3,6-tetrahydropyridinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, ditianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydro Furanyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl and 3-azabicyclo[4.1.0]heptanyl.

As used herein, the term “heteroatom” refers to one or more of oxygen, sulfur, nitrogen, phosphorus or silicon.

The term "hydroxyl" as used herein refers to the group -OH.

The term “hydroxyalkyl,” as used herein, refers to an alkyl group, as defined herein, substituted with one or more hydroxyls (hydroxyl is as defined herein). Non-limiting examples of branched or straight chain "C ₁ -C ₆ hydroxyalkyl groups include methyl, ethyl, propyl, isopropyl, isobutyl and n-butyl independently substituted with one or more hydroxyl groups. .

As used herein, the term “optionally substituted” means that the group referred to is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxyl, alkoxy, mercaptyl, cyano, halo, carbonyl , Thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, perhaloalkyl, perfluoroalkyl, and from amino, including mono and disubstituted amino groups, and protected derivatives thereof It means that it may or may not be substituted with one or more additional group(s) selected individually and independently. Non-limiting examples of optional substituents include halo, -CN, -OR, -C(O)R, -OC(O)R, -C(O)OR, -OC(O)NHR, -C(O) N(R) ₂ , -SR-, -S(=O)R, -S(=O) ₂ R, -NHR, -N(R) ₂ , -NHC(O)-, NHC(O)O- , -C(O)NH-, S(=O) ₂ NHR, -S(O) ₂ N(R) ₂ , -NHS(=O) ₂ , -NHS(O) ₂ R, C ₁ -C ₆ Alkyl, C ₁ -C ₆ alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halo-substituted C ₁ -C ₆ alkyl, halo-substituted C ₁ -C ₆ alkoxy, wherein each R is H , Halo, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halo-substituted C ₁ -C ₆ alkyl, halo-substituted C ₁ -C ₆ alkoxy Independently selected from).

As used herein, the term “affinity label” refers to a label that reversibly or irreversibly binds to another molecule.

The term “amber codon” as used herein refers to the site of incorporation of pyrrolysine, PCL and other pyrrolysine analogs, and corresponds to UAG, a nucleotide triplet in messenger RNA. The nucleotide sequence TAG is encoded in DNA and transcribed as UAG into RNA that is translated into protein. The TAG and UAG codons are used interchangeably herein to refer to the site of incorporation of pyrrolysine, PCL and other pyrrolysine analogs.

As used herein, the term “amino acid” refers to naturally occurring amino acids, non-natural amino acids, amino acid analogs, and amino acid mimetics that function in a similar manner to naturally occurring amino acids (where their structure allows for D and L stereoisomeric forms, such In all stereoisomeric forms). Amino acids are referred to herein by their name, their commonly known three letter symbols, or single letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Naturally occurring amino acids are amino acids that are encoded by the genetic code, as well as encoded amino acids that are subsequently modified. Naturally occurring amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr), Valine (Val), pyrrolysine (Pyl), selenocysteine (Sec) and pyrroline-carboxy-lysine (PCL) are included, but are not limited thereto. Modified encoded amino acids include hydroxyproline, γ-carboxyglutamate, O-phosphoserine, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-amino Butyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminobutyric acid, 3-aminobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, Desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3 -Hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, nor Valine, norleucine, ornithine, pentylglycine, pipecolic acid and thioproline are included, but are not limited thereto. The term amino acid also includes naturally occurring amino acids that, in certain organisms, are metabolites but are not encoded by the genetic code for incorporation into proteins. Such amino acids include, but are not limited to, ornithine, D-ornithine and D-arginine.

As used herein, the term “amino acid analog” refers to a compound having a basic chemical structure such as a naturally occurring amino acid, by way of example only hydrogen, a carboxyl group, an amino group and an α-carbon bonded to the R group. Amino acid analogs include natural and non-natural amino acids that are chemically blocked reversibly or irreversibly, or whose C-terminal carboxy group, their N-terminal amino group and/or their side chain functional groups are chemically modified. Such analogs include methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide, S-(carboxymethyl)-cysteine sulfone, aspartic acid-(beta-methyl ester), N-ethylglycine, alanine carboxamide, homoserine, norleucine, and methionine methyl sulfonium are included, but are not limited thereto. Certain blocking agents include, but are not limited to, t-butyloxycarbonyl (Boc) and 9-fluorenylmethyloxycarbonyl (Fmoc).

As used herein, the term “amino acid mimetic” refers to a compound that has a structure different from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.

As used herein, the term “non-natural amino acid” is intended to refer to an amino acid structure that cannot be biosynthetically produced in any organism (whether identical or different) using unmodified or modified genes from any organism. do. Additionally, it is understood that such “non-natural amino acids” require modified tRNA and modified tRNA synthetase (RS) for incorporation into proteins. These “selected” orthogonal tRNA/RS pairs are specific for non-natural amino acids and are generated by selection procedures or similar procedures developed by Schultz et al. As an example, pyrroline-carboxy-lysine is a "natural amino acid" in that it is produced biosynthetically by genes transferred from an organism to a host cell, and in that it is incorporated into proteins using natural tRNA and tRNA synthetase genes. And p-aminophenylalanine (Generation of a bacterium with a 21 amino acid genetic code, Mehl RA, Anderson JC, Santoro SW, Wang L, Martin AB, King DS, Horn DM, Schultz PG. J Am Chem. Soc. 2003 Jan 29;125(4):935-9), although produced biosynthetically, are “non-natural amino acids” because they are incorporated into proteins by “selected” orthogonal tRNA/tRNA synthetase pairs.

의 구조를 갖는 잔기를 지칭하며, 여기서 이러한 잔기는 아미노산으로부터 유래되고, R 기는 본원에 기재된 임의의 아미노산의 측쇄이다. 이러한 아미노산 잔기에는 알라니닐, 아르기니닐, 아스파라기닐, 아스파르틸, 시스테이닐, 글루타미닐, 글루타밀, 글리시닐, 히스티디닐, 이소류시닐, 류시닐, 리시닐, 메티오니닐, 페닐알라니닐, 프롤리닐, 세리닐, 트레오니닐, 트립토파닐, 티로시닐, 발리닐, 피로글루타메이트, 포르밀메티오닌, 피로글리시닐 및 셀레노시스테이닐이 포함되나, 이에 제한되지는 않는다. As used herein, the term "amino acid moiety" refers to

Refers to a residue having the structure of, wherein such residues are derived from amino acids, and the R group is the side chain of any amino acid described herein. These amino acid residues include alaninyl, arginyl, asparaginyl, aspartyl, cysteinyl, glutaminyl, glutamyl, glycinyl, histidinyl, isoleucinyl, leucinyl, lisinyl, These include methioninyl, phenylalaninyl, prolinyl, serinyl, threoninyl, tryptophanil, tyrosinyl, ballinyl, pyroglutamate, formylmethionine, pyroglycinyl and selenocysteinyl. , But is not limited thereto.

The term “amino terminal modifying group” refers to any molecule that can be attached to a terminal amino group. Such amino terminal modification groups include, but are not limited to, an amine protecting group at the end of the polymer molecule, and the polymer molecule includes, but is not limited to, polypeptides, polynucleotides and polysaccharides. Terminal modification groups also include, but are not limited to, various water soluble polymers, peptides or proteins. By way of example only, amino terminal modifying groups include polyethylene glycol or serum albumin. Certain amino terminal modifying groups are used to modify the therapeutic properties of a protein, polypeptide or peptide, for example, but not limited to, to increase the serum half-life of such a protein, polypeptide or peptide.

The term “antibody fragment” as used herein refers to any form of antibody other than the full-length form. Antibody fragments herein include antibodies, which are smaller components present in full-length antibodies, and engineered antibodies. Antibody fragments include Fv, Fc, Fab and (Fab') ₂ , single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, frames Work regions, constant regions, heavy chain, light chain, and variable regions, and alternative scaffold non-antibody molecules, bispecific antibodies, and the like. Another functional substructure is the single chain Fv (scFv) consisting of the variable regions of the immunoglobulin heavy and light chains covalently linked by a peptide linker. These small (MW <25,000 Da) proteins generally retain specificity and affinity for the antigen in a single polypeptide and can provide convenient building blocks for large antigen-specific molecules. Unless specifically stated otherwise, content and claims using the term “antibody” or “antibodies” also include “antibody fragments” and “antibody fragments”.

As used herein, the term “bioavailability” refers to the rate and extent to which a substance or active moiety thereof is delivered from a pharmaceutical dosage form and is made available at the site of action or in general circulation. An increase in bioavailability refers to an increase in the rate and extent to which a substance or an active moiety thereof is delivered from a pharmaceutical dosage form and becomes available at the site of action or in general circulation. For example, an increase in bioavailability may be indicated by an increase in the concentration of a substance or active moiety thereof in blood compared to other substances or active moieties.

As used herein, the terms "biologically active molecule", "biologically active moiety" or "biologically active" are organisms such as, but not limited to, viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals and humans. Refers to any substance capable of affecting any physical or biochemical property of a biological system, pathway, molecule or interaction with which it is associated. In particular, biologically active molecules as used herein include any substance intended for the diagnosis, cure, alleviation, treatment or prevention of a disease in humans or other animals, or otherwise improving the physical or mental well-being of a human or animal. However, it is not limited thereto. Examples of biologically active molecules include peptides, proteins, enzymes, DNA, RNA, small molecule drugs, hard drugs, soft drugs, polysaccharides, oligosaccharides, disaccharides, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleos. Seeds, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles and micelles are included, but are not limited thereto. Classes of biologically active agents suitable for use in the methods and compositions described herein include drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents. Agents, anti-anxiety agents, hormones, growth factors, steroidal agents, microvial-induced toxins, and the like.

As used herein, the term "modulation of biological activity" increases or decreases the concentration or reactivity of a protein, polypeptide, peptide, DNA, RNA, saccharide, sugar, metabolite, precursor, cofactor, or other biologically active chemical or entity. Or alter the selectivity of proteins, polypeptides, peptides, DNA, RNA, saccharides, sugars, metabolites, precursors, cofactors or other biologically active chemicals or entities, or proteins, polypeptides, peptides, DNA, RNA , A saccharide, sugar, metabolite, precursor, cofactor, or other biologically active chemical or entity.

As used herein, the term “biomaterial” refers to a biologically derived material, such as, but not limited to, a material obtained from a bioreactor and/or from recombinant methods and techniques.

The term “biophysical probe” as used herein refers to a probe that detects or monitors structural changes in a molecule by physical detection methods. Such molecules include, but are not limited to, proteins, polypeptides, peptides, DNA or RNA. Such “biophysical probes” are also used to detect or monitor the interaction of proteins, polypeptides, peptides, DNA or RNA with other molecules, such as, but not limited to, macromolecules. Examples of biophysical probes include, but are not limited to, molecular mass, nuclear spin, UV absorbance, fluorescence, circular dichroism, heat capacity, melting temperature, or other endogenous molecular properties. Examples of biophysical probes also include labels added to the molecule. Such probes include, but are not limited to, spin labels, fluorophores, isotopic labels, and photoactive groups.

As used herein, the term “biosynthetically produced” refers to any method using cells or enzymes to produce amino acids. This method involves the use of at least one of a precursor and an enzyme. In certain embodiments, such amino acids are incorporated into proteins. In certain embodiments, the biosynthesis and incorporation of amino acids occurs in the same cell, whereas in other embodiments the amino acids are produced biosynthetically in separate cells ("feeder cells") or in separate cell cultures, resulting in imanoic acid being produced in another cell. Is incorporated into the protein. In the latter case, amino acids are optionally purified from a separate cell culture, and then the purified amino acids are added to the medium of the cell culture to incorporate the amino acids into the protein.

The term “biotin analog” (also referred to as “biotin mimetic”) as used herein refers to any molecule other than biotin that binds avidin and/or streptavidin with high affinity.

The term “carboxy terminal modifying group” refers to any molecule that can be attached to a terminal carboxy group. Such carboxy terminal groups include, but are not limited to, a carboxylate protecting group at the end of the polymer molecule, and the polymer molecule includes, but is not limited to, polypeptides, polynucleotides and polysaccharides. Terminal modification groups also include, but are not limited to, various water soluble polymers, peptides or proteins. By way of example only, terminal modifying groups include polyethylene glycol or serum albumin. Certain carboxy terminal modifying groups are used to modify the therapeutic properties of proteins, polypeptides or peptides, for example, but not limited to, to increase serum half-life.

As used herein, the term “chemically cleavable group” (also referred to as “chemically labile”) refers to a group that breaks or cleaves upon exposure to an acid, base, oxidizing agent, reducing agent, chemical initiator, or radical initiator.

The term “chemiluminescent group” as used herein refers to a group that emits light as a result of a chemical reaction without applying heat. By way of example only, luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) is reacted with an _{oxidizing agent such as hydrogen peroxide (H 2} O _{2) in the presence of a base and a metal catalyst to} A state product (3-aminophthalate, 3-APA) is produced, which subsequently emits detectable light.

As used herein, the term “chromophore” refers to a molecule that absorbs light in visible, UV or IR wavelengths.

As used herein, the term “cofactor” refers to an atom or molecule essential for the functioning of a large molecule. Cofactors include, but are not limited to, inorganic ions, coenzymes, proteins, or some other factor necessary for the activity of the enzyme.

As used herein, the term “cofolding” refers to a refolding process, reaction, or method of transforming an unfolded or improperly folded molecule into an appropriately folded molecule using two or more molecules that interact with each other. By way of example only, "cofolding" uses two or more polypeptides that interact with each other to transform an unfolded or improperly folded polypeptide into an original properly folded polypeptide.

As used herein, the term “cytotoxic” refers to a compound that harms cells.

As used herein, the term “denaturing agent” or “denaturing agent” refers to any compound or substance that results in reversible unfolding of a protein. The strength of the denaturing agent or denaturing agent will be determined by both the nature and concentration of the particular denaturing agent or denaturing agent. By way of example, denaturing agents or denaturing agents include, but are not limited to, chaotropes, detergents, organic, water-miscible solvents, phospholipids, or combinations thereof. Non-limiting examples of chaotropes include, but are not limited to, urea, guanidine and sodium thiocyanate. Non-limiting examples of detergents include hard detergents such as sodium dodecyl sulfate or polyoxyethylene ether (e.g. Tween or Triton detergents), sarcosyl, mild non-ionic detergents (e.g. digitonin), Mild cationic detergents such as N-2,3-(dioleyyloxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g. sodium cholate or sodium deoxycholate) or amphoteric detergents , Such as but not limited to sulfobetaine (Zwittergent), 3-(3-chloramidopropyl)dimethylammonio-1-propane sulfate (CHAPS) and 3-(3-chloramido Propyl) dimethylammonio-2-hydroxy-1-propane sulfonate (CHAPSO). Non-limiting examples of organic, water-miscible solvents _{include, but are not limited to, acetonitrile, lower alkanols (especially C 2} -C ₄ alkanols such as ethanol or isopropanol), or lower alkanediols (C ₂ -C ₄ Alkanedols such as ethylene-glycol) can be used as denaturants. Non-limiting examples of phospholipids include, but are not limited to, naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or dipeptanoylphosphatidylcholine.

As used herein, the term "detectable label" refers to analytical techniques such as, but not limited to, fluorescence, chemiluminescence, electron-rotating resonance, ultraviolet/visible absorbance spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic Refers to a label that can be observed using resonance, radiometric and electrochemical methods.

As used herein, the term “drug” refers to any substance used in the prevention, diagnosis, alleviation, treatment or cure of a disease or condition.

As used herein, the term “dye” refers to a soluble colored material containing a chromophore.

The term “electron high density group” as used herein refers to a group that scatters electrons when irradiated with an electron beam. These groups include ammonium molybdate, bismuth subnitrate cadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate, hexamethylene tetramine, 98.5%, anhydrous indium trichloride, lanthanum nitrate, lead acetate trihydrate. , Lead citrate trihydrate, lead nitrate, periodic acid, phosphomolybdic acid, phosphotungstic acid, potassium ferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver protein compound (Ag black: 8.0-8.5%) "Stiffness ", silver tetraphenylporphine (S-TPPS), sodium chloroamate, sodium tungstate, thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranyl nitrate and vanadyl sulfate are included, It is not limited thereto.

As used herein, the term “energy transfer agent” refers to a molecule capable of donating or accepting energy from another molecule. By way of example only, fluorescence resonance energy transfer (FRET) means that the excited state energy of a fluorescent donor molecule is transferred to a recipient molecule that is not excited by non-radioactivity, and then the energy donated at a longer wavelength is emitted by fluorescence. This is a dipole-dipole coupling process.

The terms “improving” or “improving” mean increasing or prolonging the desired effect in terms of efficacy or duration. By way of example, “improving” the effect of a therapeutic agent refers to the ability to increase or prolong the effect of a therapeutic agent in terms of efficacy or duration during treatment of a disease, disorder or condition. As used herein, an “improving effective amount” refers to an amount suitable to improve the effectiveness of a therapeutic agent in the treatment of a disease, disorder or condition. When used in a patient, the amount effective for such use will depend on the severity and course of the disease, disorder or condition, the existing therapy, the patient's health status and response to the drug, and the judgment of the treating physician.

The term "eukaryotic" refers to organisms belonging to the phylogenetic domain of Eucarya, such as, but not limited to, animals (such as, but not limited to, mammals, insects, reptiles and birds), ciliated worms, plants. (Such as, but not limited to, exocotyledons, dicotyledons and algae), fungi, yeast, flagella, microspores, and substances resulting from protozoa.

The term “fatty acid” as used herein refers to a carboxylic acid having a hydrocarbon side chain _{of about C 6 or greater.}

As used herein, the term “fluorophore” refers to a molecule that emits photons upon excitation, and thus is fluorescent.

The terms "functional group", "active moiety", "activating group", "leaving group", "reactive moiety", "chemically reactive group" and "chemically reactive moiety" are somewhat synonymous in the chemical industry and perform some function or activity. And is used herein to refer to a portion of a molecule that is reactive with another molecule.

As used herein, the term “identity” refers to two or more identical sequences or subsequences. In addition, the term “substantially identical” as used herein refers to the same sequence unit when compared and aligned for maximum correspondence over a range of comparison or a designated area as measured using a comparison algorithm or by manual alignment and visual inspection. Refers to two or more sequences having a predetermined percentage. By way of example only, two or more sequences have sequence units of about 60% identity, about 65% identity, about 70% identity, about 75% identity, about 80% identity, about 85% identity, about 90 It can be "substantially identical" if it is% identity or about 95% identity. These percentages describe the "% identity" of two or more sequences. The identity of the sequence may exist over a region that is at least about 75-100 sequence units in length, over a region that is about 50 sequence units in length, or across the entire sequence unless otherwise specified. This definition also refers to the complement of the test sequence. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are identical, whereas two or more polypeptide sequences have amino acid residues of about 60% identity, about 65% identity, and about 70% identity over a specific site. , "Substantially identical" when about 75% identity, about 80% identity, about 85% identity, about 90% identity, or about 95% identity. Identity can exist over a region that is at least about 75-100 amino acids in length, over a region that is about 50 amino acids in length, or, unless otherwise specified, over the entire sequence of the polypeptide sequence. Moreover, by way of example only, two or more polynucleotide sequences are homogeneous when the nucleic acid residues are identical, whereas two or more polynucleotide sequences are about 60% identity, about 65% identity over a specific site of the nucleic acid residues, "Substantially identical" when about 70% identity, about 75% identity, about 80% identity, about 85% identity, about 90% identity, or about 95% identity. Identity can exist over a region that is at least about 75-100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or, unless otherwise specified, over the entire sequence of the polynucleotide sequence.

As used herein, the term "immunogenicity" refers to an antibody response to administration of a therapeutic drug. Immunogenicity to therapeutic proteins, polypeptides and peptides provided herein is obtained using quantitative and qualitative assays to detect antibodies to the therapeutic proteins, polypeptides and peptides in biological fluids. Such assays include, but are not limited to, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), luminescent immunoassay (LIA), and fluorescence immunoassay (FIA). Such immunogenicity assays compare the antibody response upon administration of the therapeutic proteins, polypeptides and peptides provided herein with the antibody responses upon administration of a control therapeutic protein, polypeptide or peptide or upon administration of a delivery vehicle or delivery buffer. Includes that.

As used herein, the term “intercalating agent” (also referred to as “insertable group”) refers to a molecule or group that is inserted into the intramolecular space of another molecule or the intermolecular space between molecules. By way of example only, the intercalating agent or intercalating group may be a molecule that is inserted into the lamination base of a DNA double helix.

As used herein, the term “label” refers to a substance that is incorporated into a compound and is easily detected, so that its physical distribution can be detected and/or monitored.

As used herein, the term “linkage” refers to a bond or chemical moiety formed from a chemical reaction between a functional group of a first molecule and a functional group of a second molecule. Such bonds include, but are not limited to, covalent bonds and non-covalent bonds, and such chemical moieties include esters, carbonates, imine phosphate esters, hydrazones, acetals, orthoesters, peptide linkages, oligonucleotide linkages, and Table 1 herein. Including, but not limited to, those presented in "Hydrolytically stable connection" means that the connection is substantially stable in water and does not react with water under physiological conditions for an extended period of time, such as but not limited to, at the pH value used. "Hydrolytically unstable or degradable connection" means that the connection is degradable in water or an aqueous solution, for example blood. "Enzymatically labile or degradable linkage" means that the linkage is degraded by one or more enzymes. By way of example only, certain PEGs and related polymers contain degradable linkages in the polymer backbone, or at the linking groups between the PEG polymer backbone and one or more terminal functional groups of the proteins, polypeptides or peptides provided herein. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group on a biologically active agent, and such ester groups are generally hydrolyzed under physiological conditions to obtain a biologically active agent. Release. Other hydrolytically degradable connections include carbonate connections; An imine linkage generated from the reaction of an amine and an aldehyde; A phosphate ester linkage formed by the reaction of an alcohol and a phosphate group; Hydrazone connection, which is a reaction product of hydrazide and aldehyde; An acetal connection part, which is a reaction product of an aldehyde and an alcohol; An orthoester linkage that is a reaction product of formate and alcohol; A peptide linkage formed by an amine group including, but not limited to, an amine group at the end of a polymer such as PEG, and a carboxyl group of the peptide; And a phosphoramidite group, including, but not limited to, a phosphoramidite group at the end of the polymer, and an oligonucleotide linkage formed by the 5′ hydroxyl group of the oligonucleotide, but is not limited thereto. Does not.

As used herein, the term “medium” or “medias” refers to any culture medium used to grow and harvest cells and/or products expressed and/or secreted by such cells. Such "medium" or "medium" includes any host cell, by way of example only a bacterial host cell, a yeast host cell, an insect host cell, a plant host cell, a eukaryotic host cell, a mammalian host cell, a CHO cell, a prokaryotic host. Cells, Escherichia coli, or Pseudomonas host cells, and solutions, solids, semi-solids, or rigid supports that may supplement or contain cell contents. Such “medium” or “medium” includes, but is not limited to, media or media from which the polypeptide is secreted by the growth of host cells, including, but not limited to, media before or after the proliferation step. Such "medium" or "medium" also includes, but is not limited to, a buffer or reagent containing the host cell lysate, for example, a polypeptide produced intracellularly, wherein the host cell is lysed or disrupted to release the polypeptide. Let it.

As used herein, the term “metal chelating agent” refers to a molecule that forms a complex of metal ions. By way of example, these molecules form two or more coordination bonds with the central metal ion, and optionally form a ring structure.

As used herein, the term “metal-containing moiety” refers to a group containing a metal ion, atom or particle. Such moieties include, but are not limited to, cisplatin, chelated metal ions (eg nickel, iron and platinum) and metal nanoparticles (eg nickel, iron and platinum).

As used herein, the term "moiety incorporating a heavy atom" refers to a group incorporating an ion of an atom heavier than carbon. Such ions or atoms include, but are not limited to, silicon, tungsten, gold, lead, and uranium.

As used herein, the term “modified” refers to the presence of a change to an amino acid or amino acid residue, and such change or modification is obtained by chemical or biochemical processes.

As used herein, the term "regulated serum half-life" refers to a positive or negative change in the circulating half-life of a modified biologically active molecule compared to its unmodified form. By way of example, modified biologically active molecules include, but are not limited to, compounds provided herein. As an example, serum half-life is measured by taking a blood sample at various time points after administration of a biologically active molecule or a modified biologically active molecule, and determining the concentration of that molecule in each sample. The correlation between serum concentration and time can be used to calculate the serum half-life. As an example, a controlled serum half-life can be an increase in serum half-life, which enables improved dosing regimens or avoids toxic effects. This increase in serum may be at least about 2 times, at least about 3 times, at least about 5 times, or at least about 10 times.

As used herein, the term “nanoparticle” refers to a particle having a particle size of about 500 nm to about 1 nm.

As used herein, the term “approximate-stoichiometric” refers to a molar ratio of compounds participating in a chemical reaction between about 0.75 and about 1.5.

As used herein, the term “non-eukaryotic cell” refers to a non-eukaryotic organism. For example, non-eukaryotic organisms belong to the eubacterial phylogenetic domain, including Escherichia coli, Thermus thermophilus (Thermus thermophilus ), or Bacillus stearomophilus stearothermophilus ), Pseudomonas fluorescens , Pseudomonas aeruginosa ( Pseudomonas) aeruginosa ), Pseudomonas putida ) is included, but not limited thereto, or belongs to the archaeal phylogenetic domain, including metanococcus jannaschii , metanobacterium thermoautotrophicum (Methanobacterium thermoautotrophicum), arcaeoglobus Archaeoglobus fulgidus ), Pyrococcus furiosus ), Pyrococcus horikoshii , Aeuropyrum pernix ), or halobacterium, such as Haloferax volcanii (Haloferax volcanii ) and Halobacterium species NRC-1 are included, but are not limited thereto.

The term “nucleic acid” as used herein refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single- or double-stranded form. By way of example only, such nucleic acids and nucleic acid polymers include (i) analogs of natural nucleotides having properties similar to those of a reference nucleic acid; (ii) oligonucleotide analogs such as, but not limited to, PNA (peptidonucleic acid), analogs of DNA used in antisense technology (phosphorothioate, phosphoroamidate, etc.); (iii) Conservatively modified variants thereof (such as, but not limited to, degenerate codon substitutions) and complementary sequences and clearly indicated sequences. By way of example, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and/or deoxyinosine residue.

As used herein, the term “oxidizing agent” refers to a compound or substance capable of removing electrons from the compound to be oxidized. By way of example, oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythritol, and oxygen. A variety of oxidizing agents are suitable for use in the methods and compositions described herein.

As used herein, the term “photoaffinity label” refers to a label having a group that upon exposure to light, the label forms a linkage with an affinity molecule. By way of example only, these connections can be shared or non-shared.

As used herein, the term “photocausing moiety” refers to a group that binds covalently or non-covalently with an ion or other molecule upon light emission at a specific wavelength.

As used herein, the term “photodegradable group” refers to a group that is destroyed upon exposure to light.

As used herein, the term “photocrosslinking agent” refers to a compound that is reactive upon exposure to light and comprises two or more functional groups that form covalent or non-covalent bonds with two or more monomeric or polymeric molecules.

The term “photoisomerizing moiety” as used herein refers to a group that changes from one isomeric form to another upon luminescence by light.

As used herein, the term “polyalkylene glycol” refers to a linear or branched polymeric polyether polyol. Such polyalkylene glycols include, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof.

As used herein, the term “polymer” refers to a molecule made up of repeated subunits. Such molecules include, but are not limited to, proteins, polypeptides, peptides, polynucleotides or polysaccharides or polyalkylene glycols.

The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. That is, the description of the polypeptide applies equally to the description of the peptide and the description of the protein, and vice versa. Amino acid residues include those derived from natural and unnatural amino acids. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are compounds provided herein. Additionally, such “polypeptide”, “peptide” and “protein” include amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds or other linkages.

The term “post-translational modified” refers to any modification of an amino acid that occurs after the amino acid has been incorporated into a polypeptide chain by translation. Such modifications include, but are not limited to, post-translational in vivo modifications and post-translational in vitro modifications.

As used herein, the term “protected” refers to the presence of a “protecting group” or moiety that prevents reaction of a chemically reactive functional group under certain reaction conditions. The protecting group will depend on the type of chemically reactive group to be protected. By way of example only, (i) when the chemically reactive group is an amine or hydrazide, the protecting group may be selected from tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) when the chemically reactive group is thiol, the protecting group may be orthopyridyldisulfide; (iii) When the chemically reactive group is a carboxylic acid such as butanoic acid or propionic acid, or a hydroxyl group, the protecting group may be benzyl or an alkyl group such as methyl, ethyl or tert-butyl. By way of example only, the blocker/protecting group is also acetamide, allyloxycarbonyl, allyl ether, benzyl, benzylamine, benzylideneamine, benzyl carbamate, benzyl ester, methyl ester, t-butyl ester, St-butyl Ester, 2-alkyl-1,3-oxazoline, dimethyl acetal, 1,3-dioxane, 1,3-ditian, N,N-dimethylhydrazone, phthalimide, trityl, paramethoxybenzyl ether , Carbobenzyloxy, p-toluenesulfonamide, trifluoroacetamide, triphenylmethylamine, t-butyl ether, benzyl ether, t-butyldimethylsilyl ether, t-butyldiphenylsilyl ether, 2-(trimethyl Silyl) ethoxycarbonyl, acetic acid ester, pivalic acid ester, benzoic acid ester, acetonide, benzylidene acetal, and photodegradable groups such as Nvoc and MeNvoc.

As used herein, the term “radioactive moiety” refers to a group in which the nucleus spontaneously emits nuclear radiation, such as alpha or beta particles, or gamma radiation.

As used herein, the term “reactive compound” refers to a compound that is reactive toward another atom, molecule or compound under appropriate conditions.

The term “recombinant host cell” (also referred to as “host cell”) refers to a cell comprising an exogenous polynucleotide, wherein the method used to insert the exogenous polynucleotide into the cell includes direct uptake, transformation, f-crossing, or Other methods known in the art for the generation of recombinant host cells include, but are not limited to. By way of example only, such exogenous polynucleotides may be unincorporated vectors, such as, but not limited to, plasmids, or may be incorporated into the host genome.

The term "redox-active agent" as used herein refers to a molecule by which the redox active agent is reduced or oxidized by oxidizing or reducing another molecule. Examples of redox activators include, but are not limited to, ferrocene, quinone, Ru ^{2 +/3+} complex, Co ^{2 +/3+} complex, and Os ^{2 +/3+ complex.}

As used herein, the term “reducing agent” refers to a compound or substance capable of adding electrons to the compound to be reduced. By way of example, reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol) and reduced glutathione. By way of example only, such reducing agents can be used to maintain sulfhydryl groups in the reduced state and to reduce intramolecular or intermolecular disulfide bonds.

As used herein, the term “refolding” describes any process, reaction or method that transforms an improperly folded or unfolded state into its original or properly folded form. By way of example only, with respect to disulfide bonds, refolding is to modify the disulfide bond containing a protein or polypeptide from an improperly folded or unfolded state to its original or properly folded form. Such disulfide bonds containing proteins or polypeptides include proteins or polypeptides into which the compounds provided herein have been incorporated. Refolding is often initiated by the removal of chaotropic agents, such as urea or guanidinium hydrochloride, previously added to the protein solution for solubilization and unfolding of the protein.

The term “resin” as used herein refers to high molecular weight, insoluble polymer beads. By way of example only, these beads can be used as a support for solid-phase peptide synthesis or as a site for attachment of molecules prior to purification.

As used herein, the term “saccharide” refers to a series of carbohydrates, such as, but not limited to, sugars, monosaccharides, oligosaccharides and polysaccharides.

As used herein, the term “spin label” is an atom or group of atoms that exhibits unpaired electron spin (ie, a stable paramagnetic group) that can be detected by electron spin resonance spectroscopy and attached to another molecule. Refers to a molecule containing. Such spin-labeled molecules include, but are not limited to, nitrile radicals and nitroxides, and may be single spin labels or double spin labels.

As used herein, the term “stoichiometric” refers to the molar ratio of the compounds participating in the chemical reaction being about 0.9 to about 1.1.

As used herein, the term “substantially purified” refers to an ingredient of interest that is substantially or essentially free of other ingredients that normally accompany or interact with the ingredient of interest prior to purification. By way of example only, the formulation of the component of interest is less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%. , If it contains less than about 2% or less than about 1% (dry weight) of the contaminating component, the component of interest may be “substantially purified”. Thus, the "substantially purified" component of interest is about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%. Or it may have a purity level greater than that. By way of example only, a protein, polypeptide or peptide containing a compound provided herein is purified from the original cell or host cell. By way of example only, preparations of proteins, polypeptides or peptides containing the compounds provided herein are less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%. , Less than about 4%, less than about 3%, less than about 2% or less than about 1% (by dry weight) of contaminants, the preparation is "substantially purified". By way of example only, when a protein, polypeptide or peptide containing a compound provided herein is recombinantly produced by a host cell, the protein, polypeptide or peptide containing the compound provided herein will be about 30% of the dry weight of the cell. %, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less. By way of example only, when a protein, polypeptide or peptide containing a compound provided herein is recombinantly produced by a host cell, the protein, polypeptide or peptide containing the compound provided herein is about 5 g/g/peptide in the culture medium. L, about 4 g/L, about 3 g/L, about 2 g/L, about 1 g/L, about 750 mg/L, about 500 mg/L, about 250 mg/L, about 100 mg/L, About 50 mg/L, about 10 mg/L or about 1 mg/L or less. By way of example only, "substantially purified" proteins, polypeptides or peptides containing the compounds provided herein are suitable methods including, but not limited to, SDS/PAGE analysis, RP-HPLC, SEC and capillary electrophoresis. As measured by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85 %, about 90, about 95%, about 99% or more.

As used herein, the term “toxic moiety” refers to a compound that can cause harm or death.

The terms “treat”, “treating” or “treatment” as used herein alleviate, reduce or ameliorate symptoms of a disease or condition, prevent further symptoms, or improve or prevent underlying metabolic causes of symptoms, or , Inhibiting a disease or condition, for example, arresting the progression of the disease or condition, alleviating the disease or condition, regressing the disease or condition, alleviating the condition caused by the disease or condition, or It includes stopping the symptoms of the disease or condition. The terms “treat”, “treating” or “treatment” include, but are not limited to, prophylactic and/or curative treatment.

As used herein, the term “water-soluble polymer” refers to any polymer that is soluble in an aqueous solvent. Such water-soluble polymers include polyethylene glycol, polyethylene glycol propionaldehyde, mono C ₁ -C ₁₀ alkoxy or aryloxy derivatives thereof, monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic acid. Anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives such as dextran sulfate, polypropylene glycol, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol, Heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives such as methylcellulose and carboxymethyl cellulose, etc., serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycols and derivatives thereof, polyalkyl Copolymers of ene glycol and derivatives thereof, polyvinyl ethyl ether and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, etc., or mixtures thereof. By way of example only, by coupling of such a water soluble polymer with a protein, polypeptide or peptide containing a compound provided herein, increased water solubility, increased or regulated serum half-life, increased or controlled therapeutic half-life compared to the unmodified form. , Increased bioavailability, regulated biological activity, prolonged circulation time, regulated immunogenicity, regulated physical association properties, such as aggregation and multimer formation, altered receptor binding, altered binding with one or more binding partners, and altered receptors. Changes occur, including, but not limited to, dimerization or multimerization and the like.

Other objects, features, and advantages of the methods, compositions and combinations described herein will become apparent from the detailed description below. However, it should be understood that although this detailed description and specific examples represent specific embodiments, they are provided by way of example only.

Biosynthetically Produced pyrrolysine and PCL Site-specific incorporation of

Pyrrolysine (PYL) is the 22nd natural genetically encoded amino acid found in certain methanogenic archaea and two unrelated bacterial species of the Methanosarcinaceae family. Specifically, pyrrolysine is found in MtmB1, a monomethylamine (MMA) methyltransferase that initiates methane formation in these archaea bacteria (Srinivasan, G., James, CM, and Krzycki, JA (2002), " Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA," Science , 296 , 1459-62]; [Soares, JA, Zhang, L., Pitsch, RL, Kleinholz, NM, Jones, RB, Wolff, JJ, Amster, J., Green-Church, KB, and Krzycki, JA (2005), "The residue mass of L-pyrrolysine in three distinct methylamine methyltransferases," Journal of Biological Chemistry , 280 , 36962-9]; [Hao, B., Gong, W., Ferguson, TK, James, CM, Krzycki, JA, and Chan, MK, (2002), "A new UAG-encoded residue in the structure of a methanogen methyltransferase", Science , 296 , 1462-6]; [Krzycki, JA, (2005), "The direct genetic encoding of pyrrolysine," Current Opinion in Microbiology , 8 , 706-12]; [Krzycki, JA, (2004), "Function of genetically encoded pyrrolysine in corrinoid-dependent methylamine methyltransferases," Current Opinion in Chemical Biology , 8 , 484-91], and [Ambrogelly, A., Palioura, S., and Soll, D., (2007), "Natural expansion of the genetic code," Nature Chemical Biology 3 , 29-35). Pyrrolysine is believed to be a dipeptide in which the ε-amine of lysine is linked to the D-isomer of 4-methyl-pyrroline-5-carboxylate via an amide bond (Polycarpo, CR, Herring, S., Berube, A., Wood, JL, Soll, D., and Ambrogelly, A., (2006), "Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase," FEBS Letters , 580 , 6695-700). The structure of pyrrolysine (Fig. 1) was deduced from the crystal structure of MtmB1 and the mass of the moiety (J. Biol. Chem. 2005, 44, 36962-36969]; see [PNAS, 2007, 104, 1021-1026]. ).

The mtmB1 gene encoding MtmB1 normally has an in-frame amber (TAG) codon, which is a reference stop codon. However, in mtmB1 mRNA, the UAG codon encoded as TAG at the DNA level does not terminate translation during production of the MtmB1 protein, but instead the UAG codon encodes for pyrrolysine incorporated into the protein. Pyrrolysine is synthesized endogenously and then incorporated into this in-frame UAG codon as a free amino acid by simultaneous translation.

Pyrrolysine is readily biosynthesized and incorporated by the natural genes pylT, pylS, pylB, pylC and pylD. pylT encodes pyrrolysyl-tRNA, pylS encodes pyrrolysyl-tRNA synthetase, while pylB, pylC and pylD encode proteins required for the biosynthesis of pyrrolysine. These genes are derived from Methanosarcina mazei (Longstaff, DG, Larue, RC, Faust, JE, Mahapatra, A., Zhang, L., Green-Church, KB, and Krzycki, JA , (2007), "A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine," Proceedings of the National Academy of Sciences of the United States of America , 104 , 1021-6], and [Namy, O., Zhou, Y., Gundllapalli, S., Polycarpo, CR, Denise, A., Rousset, JP, Soll, D., and Ambrogelly, A., (2007), "Adding pyrrolysine to the Escherichia coli genetic code," FEBS Letters , 581 , 5282-8). The pylT and pylS genes together with the pylB, pylC and pylD genes form the pylTSBCD gene cluster, which is a natural genetic coding expansion cassette, and its delivery is pyrrolysine in which the UAG codon is incorporated into the protein at the UAG site (endogenously synthesized Free amino acids).

The biosynthesis of pyrrolysine has been suggested as being facilitated by the gene products of the natural genes pylB, pylC and pylD along with D-ornithine proposed as a precursor (Namy, O., Zhou, Y., Gundllapalli, S., Polycarpo, CR, Denise, A., Rousset, JP, Soll, D., and Ambrogelly, A., (2007), "Adding pyrrolysine to the Escherichia coli genetic code," FEBS Letters , 581 , 5282-8). Various precursors for pyrrolysine, such as D-glutamate, D-isoleucine, D-proline and D-ornithine, have been proposed, but D-ornithine is by a plasmid with the natural genes pylT, pylS, pylB, pylC and pylD. It has been proposed to be the most effective precursor for pyrrolysine biosynthesis in transformed Escherichia coli. This study used translating in TAG incorporation or break signal, i.e., the production of full-length proteins, and pyrrolysine in Escherichia coli transformed by a plasmid with the natural genes pylT, pylS, pylB, pylC and pylD. It was demonstrated that it was biosynthesized and incorporated into the produced protein using D-ornithine as a precursor. The incorporation of pyrrolysine was not directly verified by mass spectrometry, but the conclusion is that plasmids with the natural genes pylT, pylS, pylB, pylC and pylD although low levels of pyrrolysine in the absence of added D-ornithine. It was supported by previous mass spectrometry data in that it was biosynthesized and incorporated into proteins produced in Escherichia coli transformed by (Longstaff, DG, Larue, RC, Faust, JE, Mahapatra, A. , Zhang, L., Green-Church, KB, and Krzycki, JA, (2007), "A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine," Proceedings of the National Academy of Sciences of the United States of America , 104 , 1021-6).

However, as provided herein, the incorporation of the pylT, pylS, pylB, pylC and pylD genes into Escherichia coli or mammalian cells, as identified using mass spectrometry, and D-ornis into the growth medium. It was found that by the addition of tin, “demethylated pyrrolysine” was biosynthesized and incorporated (Fig. 1, PCL-A and PCL-B). This observation is surprising and unpredictable in light of the results proposed by Longstaff et al. Thus, using the natural genes pylT, pylS, pylB, pylC and pylD and D-ornithine as a precursor, a pyrrolysine analog, pyrroline-carboxy-, which is naturally encoded, produced biosynthetically, and incorporated into a protein. Lysine (PCL) is provided herein. In another embodiment, since D-arginine is a precursor to D-ornithine, it is naturally encoded and produced biosynthetically, using the natural genes pylT, pylS, pylB, pylC and pylD and D-arginine as a precursor. Thus, pyrrolysine analogs incorporated into proteins are also provided herein.

Initially, the formation of the pyrroline ring of pyrrolysine from D-ornithine was thought to be similar to that of proline from L-ornithine (Longstaff, DG, Larue, RC, Faust, JE, Mahapatra, A., Zhang, L., Green-Church, KB, and Krzycki, JA, (2007), "A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine," Proceedings of the National Academy of Sciences of the United States of America , 104 , 1021-6). Numerous bacteria convert L-ornithine to L-proline via the 1-pyrroline-5-carboxylate intermediate. The formation of 1-pyrroline-5-carboxylate from ornithine is the first step in the two-step process in the biosynthesis of L-proline. The formation of 1-pyrroline-5-carboxylate from L-ornithine can occur via two pathways, the first pathway being 1-pyrroline-5-car by cyclodeamination of L-ornithine. It is the formation of boxylate, and the second pathway is the formation of L-glutamate semialdehyde by L-ornithine amino transferase. Subsequently, glutamate semialdehyde forms 1-pyrroline-5-carboxylate. L-proline is formed by reduction of 1-pyrroline-5-carboxylate with pyrroline-5-carboxylate reductase.

Recently, no homology with L-ornithine amino transferase was confirmed in the formation of pyrrolysine in methanogenic archaea. However, it is expected to be related to similar enzymes. The biosynthesis of pyrrolysine is thought to be a consistent process using cell precursors and a consistent action of the products of the pylB, pylC and pylD genes, and biosynthesis of pyrrolysine from D-proline via 1-pyrroline-5-carboxylate. The proposed scheme for this is shown in Figure 2A (Longstaff, DG, Larue, RC, Faust, JE, Mahapatra, A., Zhang, L., Green-Church, KB, and Krzycki, JA, (2007). ), "A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine," Proceedings of the National Academy of Sciences of the United States of America , 104 , 1021-6). The pylB, pylD and pylD gene products are members of several protein families. PylB has a signature residue of the family of Fe-S radical SAM enzymes known to mediate radical-catalyzed reactions. In addition, PylB shares some sequence homology with biotin synthase, so it has been suggested that PylB is involved in the formation or methylation of the pyrrolysine pyrroline ring. The pylD gene product, PylD, has a NADH-binding domain of several families of dehydrogenases, suggesting that PylD is involved in the formation of 1-pyrroline imine bonds. PylC is similar to carbamoylphosphate synthetase and D-alanyl-D-alanine ligase, which is a pyrrolysine amide between lysine and the D-isomer of 4-methyl-1-pyrroline-5-carboxylate. Suggests a role in the formation of bonds Thus, the putative pylB, pylC and pylD gene products have respective similarities to the radical SAM protein, protein forming amino acid and amino acid dehydrogenase, and are therefore thought to participate in similar pathways for the biosynthesis of pyrrolysine.

However, as provided herein, an attempt to biosynthesize pyrrolysine in Escherichia coli and HEK293F cells using D-ornithine as a precursor does not form pyrrolysine, but rather “demethylated pyrrolysine” (herein Pyrroline-carboxy-lysine (PCL) (also referred to as PCL-A or PCL-B: see FIG. 1) is formed. As described herein, the biosynthesis of PCL-A or PCL-B does not require the presence of the pylB gene, so a possible scheme for biosynthesis of PCL-A or PCL-B is shown in Figure 2B. While not wishing to be bound by any particular theory, this possible pathway is the conversion of D-ornithine to 5-amino-2-oxopentanoic acid via endogenous D-amino acid oxidase (EC 1.4.3.3), and 1-pi. And spontaneous cyclization of 5-amino-2-oxopentanoic acid with roline-2-carboxylate and water. This precursor, available in most organisms, can be converted to D-1-pyrroline-5-carboxylate, possibly by rearrangement of the double bond with the aid of the enzyme PylD. L-lysine to epsilon amine ligation of D-1-pyrroline-5-carboxylate by PylC and ATP will yield pyrroline-carboxy-lysine (PCL: PCL-A). Alternatively, ligation of 1-pyrroline-2-carboxylate can result in PCL-B having the same molecular weight as PCL-A. The observation that both PylD and PylC are required for the incorporation of PCL, and that PylS is not acceptable as a substrate pyrrolysine analog with a sp2 carbon at a position equivalent to the C-5 position of the 1-pyrroline ring of PCL (here As shown in), it is suggested that in the beginning, PCL can be incorporated into proteins mainly in the form of PCL-A. While not wishing to be bound by any particular theory, the demethylated PCL-A or PCL-B is necessary cofactor(s) in the presence of D-ornithine added as a result of inactivation of PylB, in the absence of a methyl donating PylB substrate, ) In the absence of or in a combination thereof. However, this does not preclude the existence of alternative mechanisms. Indeed, incorporation tests with several intermediates (as presented herein) suggest a different biosynthetic pathway for PCL-A or PCL-B than that suggested in Figure 2B. This alternative route is described herein.

Incorporation of pyrrolysine into the protein in response to the UAG codon is facilitated by the natural genes pylT and pylS, where UAG translation as pyrrolysine is amber into pyrrolysine by pyrrolysyl-tRNA synthetase (PylS). It was confirmed that the decoding tRNA ^pyl requires aminoacylation (Fig. 3A). The pylT gene encodes a specific natural inhibitor tRNA ^pyl (PylT), whose CUA anticodon is complementary to the UAG sense codon corresponding to pyrrolysine. The pylS gene encodes a specific class II tRNA synthetase, pyrrolysyl-tRNA synthetase (PylS), which ^{confers pyrrolysine} (chemically or biochemically synthesized) to the tRNA pyl, and also pyrrolysine- A dependent ATP: pyrophosphate exchange reaction is carried out. Similarly, incorporation of the pyrrolysine analogue PCL-A or PCL-B into the protein in response to the UAG codon is thought to be facilitated by the natural genes pylT and pylS. (Figure 3B).

Biosynthesis and site-specific incorporation of pyrrolysine and PCL into proteins expressed by prokaryotic and eukaryotic cells

Biosynthetically produced pyrrolysine and/or pyrroline-carboxy-lysine ((S)-2-amino-6-(3,4-dihydro-2H-pyrrole-2-carboxamido)hexanoic acid ( Method for site specific incorporation of PCL-A) or (S)-2-amino-6-(3,4-dihydro-2H-pyrrole-5-carboxamido)hexanoic acid (PCL-B)) Is provided herein. The pyrrolysine analogs PCL-A and PCL-B (both referred to herein as PCL) lack the methyl group of pyrrolysine (PYL) (FIG. 1 ). In certain embodiments of this method, the eukaryotic cell is a mammalian cell, a yeast cell, an insect cell, a fungal cell or a plant cell. In other embodiments, the mammalian cells used in the methods provided herein include human embryonic kidney (HEK293F) cells, human epithelial carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamsters. Kidney (BHK-21) cells and Chinese hamster ovary (CHO) cells. In certain embodiments, yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells used in the methods provided herein include Spodoptera frugiperda (Spodoptera frugiperda ) (sf9 and sf21) cells, Trichoplusia ni (BTI TN-5B1-4 or High-Five™) cells and Mammestra brassicae brassicae ) cells are included, but are not limited thereto. In certain embodiments, the prokaryotic cells are bacteria, while in other embodiments, the bacteria used in the methods provided herein include Escherichia coli, Mycobacterium smegmatis. smegmatis ), Lactococcus lactis ) and Bacillus subtilis .

: (2S)-2-아미노-6-(2,5-디아미노펜탄아미도)헥산산이다. 특정 실시양태에서, 전구체는

: (2S)-2-아미노-6-((R)-2,5-디아미노펜탄아미도)헥산산이다. 특정 실시양태에서, 전구체는

: 2,5-디아미노-3-메틸펜탄산이다. 특정 실시양태에서, 전구체는 (2R,3R)-2,5-디아미노-3-메틸펜탄산이다. 특정 실시양태에서, 진핵 세포는 포유동물 세포, 효모 세포, 곤충 세포, 진균 세포 또는 식물 세포이다. 다른 실시양태에서, 본원에 제공된 방법에 사용된 포유동물 세포에는 인간 배아 신장 HEK293F 세포, 인간 상피양 암종 HeLa 및 GH3 세포, 원숭이 신장 COS 세포, 래트 C6 신경교종 세포, 새끼 햄스터 신장 BHK-21 세포 및 차이니즈 햄스터 난소 CHO 세포가 포함되나, 이에 제한되지는 않는다. 특정 실시양태에서, 본원에 제공된 방법에 사용된 효모 세포에는 사카로미세스 세레비지애 및 피키아 파스토리스 세포가 포함되나, 이에 제한되지는 않는다. 다른 실시양태에서, 본원에 제공된 방법에 사용된 곤충 세포에는 스포도프테라 프루기페르다 sf9 및 sf21 세포, 트리코플루시아 니 (BTI TN-5B1-4 또는 하이-파이브™) 세포 및 맘메스트라 브라시카에 세포가 포함되나, 이에 제한되지는 않는다. 특정 실시양태에서, 원핵 세포는 박테리아이고, 한편 다른 실시양태에서, 본원에 제공된 방법에 사용된 박테리아에는 에스케리키아 콜라이, 미코박테리움 스메그마티스, 락토코쿠스 락티스 및 바실러스 서브틸리스가 포함되나, 이에 제한되지는 않는다. In certain embodiments, such methods for site-specific incorporation of biosynthetically generated pyrrolysine and PCL include the genes pylT, pylS, pylB, pylC and pylD, and the genes for the desired protein in prokaryotic and/or eukaryotic cells. And optionally adding pyrrolysine or a precursor for PCL to the growth medium of the transfected cells. In certain embodiments, the precursor is D-ornithine, while in other embodiments the precursor is L-ornithine. In certain embodiments, the precursor is D,L-ornithine. In certain embodiments, the precursor is D-arginine, while in other embodiments the precursor is L-arginine. In certain embodiments, the precursor is D,L-arginine. In certain embodiments, the precursor is

: (2S)-2-Amino-6-(2,5-diaminopentanamido)hexanoic acid. In certain embodiments, the precursor is

: (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid. In certain embodiments, the precursor is

: It is 2,5-diamino-3-methylpentanoic acid. In certain embodiments, the precursor is (2R,3R)-2,5-diamino-3-methylpentanoic acid. In certain embodiments, the eukaryotic cell is a mammalian cell, yeast cell, insect cell, fungal cell or plant cell. In other embodiments, the mammalian cells used in the methods provided herein include human embryonic kidney HEK293F cells, human epithelial carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-21 cells, and Chinese hamster ovary CHO cells are included, but are not limited thereto. In certain embodiments, yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells used in the methods provided herein include Spodoptera frugiperda sf9 and sf21 cells, Trichoflusia ni (BTI TN-5B1-4 or High-Five™) cells, and Mammestra brassica. Including, but not limited to, cells. In certain embodiments, the prokaryotic cells are bacteria, while in other embodiments, bacteria used in the methods provided herein include Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis, and Bacillus subtilis. , But is not limited thereto.

In certain embodiments, such methods for site-specific incorporation of biosynthetically generated pyrrolysine and PCL include the genes pylT, pylS, pylB, pylC and pylD, and the genes for the desired protein in prokaryotic and/or eukaryotic cells. Incorporating into cells and adding pyrrolysine or a precursor for PCL to the growth medium of the transfected cells. In certain embodiments, the precursor is D-ornithine, while in other embodiments the precursor is L-ornithine. In certain embodiments, the precursor is D,L-ornithine. In certain embodiments, the precursor is D-arginine, while in other embodiments the precursor is L-arginine. In certain embodiments, the precursor is D,L-arginine. In certain embodiments, the precursor is (2S)-2-amino-6-(2,5-diaminopentanamido)hexanoic acid. In certain embodiments, the precursor is (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid. In certain embodiments, the precursor is 2,5-diamino-3-methylpentanoic acid. In certain embodiments, the precursor is (2R,3R)-2,5-diamino-3-methylpentanoic acid. In certain embodiments, the eukaryotic cell is a mammalian cell, yeast cell, insect cell, fungal cell or plant cell. In other embodiments, the mammalian cells used in the methods provided herein include human embryonic kidney HEK293F cells, human epithelial carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-21 cells, and Chinese hamster ovary CHO cells are included, but are not limited thereto. In certain embodiments, yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, insect cells used in the methods provided herein include Spodoptera frugiperda sf9 and sf21 cells, Trichoflusia ni (BTI TN-5B1-4 or High-Five™) cells, and Mammestra brassica. Including, but not limited to, cells. In certain embodiments, the prokaryotic cells are bacteria, while in other embodiments, bacteria used in the methods provided herein include Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis, and Bacillus subtilis. , But is not limited thereto.

In certain embodiments, such a method for site-specific incorporation of biosynthetically generated PCL comprises incorporating the genes pylT, pylS, pylC and pylD, and genes for the desired protein into prokaryotic and/or eukaryotic cells and growing It involves adding D-ornithine as a precursor for PCL to the medium. In certain embodiments, the eukaryotic cell is a mammalian cell, yeast cell, insect cell, fungal cell or plant cell. In other embodiments, the mammalian cells used in the methods provided herein include human embryonic kidney HEK293F cells, human epithelial carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-21 cells, and Chinese hamster ovary CHO cells are included, but are not limited thereto. In certain embodiments, yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, insect cells used in the methods provided herein include Spodoptera frugiperda sf9 and sf21 cells, Trichoflusia ni (BTI TN-5B1-4 or High-Five™) cells, and Mammestra brassica. Including, but not limited to, cells. In certain embodiments, the prokaryotic cells are bacteria, while in other embodiments, bacteria used in the methods provided herein include Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis, and Bacillus subtilis. , But is not limited thereto. In this embodiment in which the natural genes pylT, pylS, pylC and pylD are used, rather than pyrrolysine produced and incorporated biosynthetically, instead the demethylated pyrrolysine analogue PCL is produced and incorporated biosynthetically.

In certain embodiments, such methods for site-specific incorporation of biosynthetically generated pyrrolysine and PCL include the genes pylT, pylS, pylC and pylD, and the genes for the desired protein into prokaryotic and/or eukaryotic cells. D-ornithine, L-ornithine, D,L-ornithine, D-arginine, L-arginine, D,L-arginine, (2S )-2-amino-6-(2,5-diaminopentanamido)hexanoic acid, (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid or And adding 2,5-diamino-3-methylpentanoic acid or (2R,3R)-2,5-diamino-3-methylpentanoic acid. In certain embodiments, the eukaryotic cell is a mammalian cell, yeast cell, insect cell, fungal cell or plant cell. In other embodiments, the mammalian cells used in the methods provided herein include human embryonic kidney HEK293F cells, human epithelial carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-21 cells, and Chinese hamster ovary CHO cells are included, but are not limited thereto. In certain embodiments, yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, insect cells used in the methods provided herein include Spodoptera frugiperda sf9 and sf21 cells, Trichoflusia ni (BTI TN-5B1-4 or High-Five™) cells, and Mammestra brassica. Including, but not limited to, cells. In certain embodiments, the prokaryotic cells are bacteria, while in other embodiments, bacteria used in the methods provided herein include Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis, and Bacillus subtilis. , But is not limited thereto. In this embodiment in which the natural genes pylT, pylS, pylC and pylD are used, rather than pyrrolysine produced and incorporated biosynthetically, instead the demethylated pyrrolysine analogue PCL is produced and incorporated biosynthetically.

In certain embodiments, such a method for site-specific incorporation of biosynthetically generated pyrrolysine incorporates the genes pylT, pylS, pylC and pylD, and genes for the desired protein into prokaryotic and/or eukaryotic cells, It involves adding 2,5-diamino-3-methylpentanoic acid or (2R,3R)-2,5-diamino-3-methylpentanoic acid as a precursor for pyrrolysine to the growth medium. In certain embodiments, the eukaryotic cell is a mammalian cell, yeast cell, insect cell, fungal cell or plant cell. In other embodiments, the mammalian cells used in the methods provided herein include human embryonic kidney HEK293F cells, human epithelial carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-21 cells, and Chinese hamster ovary CHO cells are included, but are not limited thereto. In certain embodiments, yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, insect cells used in the methods provided herein include Spodoptera frugiperda sf9 and sf21 cells, Trichoflusia ni (BTI TN-5B1-4 or High-Five™) cells, and Mammestra brassica. Including, but not limited to, cells. In certain embodiments, the prokaryotic cells are bacteria, while in other embodiments, bacteria used in the methods provided herein include Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis, and Bacillus subtilis. , But is not limited thereto.

The site-specific incorporation of PCL biosynthetically generated at the TAG-encoded site of the model protein (hRBP4) uses the natural genes pylT, pylS, pylB, pylC and pylD, and D-ornithine added to the growth medium as a precursor. And carried out in mammalian cells (see Example 2). In one embodiment, the site-specific incorporation of PCL into the model protein, hRBP4, transforms HEK293F cells into UAG-recognized tRNA PylT, its specific aminoacyl-tRNA synthetase PylS, biosynthetic genes pylB, pylC and DNA encoding pylD, as well as This was achieved by co-transfection with DNA encoding the target protein with the incorporation site encoded by the TAG. Another gene construct used for this in vivo biosynthesis and incorporation in mammalian cells into the model protein hRBP4 at a single site specified by the TAG codon is shown in FIG. 4A. Other gene constructs for use in mammals for the expression of various single site TAG mutants of the Fc domain of hRBP4, mEPO, hEPO, and mIgG1 are shown in Figure 4B. Plasmids having pylT, pylS, pylB, pylC and pylD for the incorporation of PCL or pyrrolysine biosynthetically induced in Escherichia coli cells are shown in FIG. 5.

When D-ornithine, a putative precursor for the biosynthesis of pyrrolysine, is added to the culture medium of HEK293F cells transfected with DNA encoding the target protein as well as pylT, pylS, pylB, pylC and pylD, not pyrrolysine but PCL It is incorporated into hRBP4 at the site of this TAG codon (see Example 2, Fig. 6A). The incorporation efficiency depends on the different incorporation sites as shown in Fig. 6A.

The site-specific incorporation of PCL biosynthetically generated at the TAG-encoded site of the model protein (hRBP4) was performed using the natural genes pylT, pylS, pylC and pylD, and D-ornithine added to the growth medium as a precursor. It was carried out in cells (see Example 2). In one embodiment, the site-specific incorporation of PCL into the model protein, hRBP4, transforms HEK293F cells into UAG-recognized tRNA PylT, its specific aminoacyl-tRNA synthetase PylS, DNA encoding the biosynthetic genes pylC and pylD, as well as TAG. It was achieved by co-transfection with DNA encoding the target protein with the incorporation site encoded by. When D-ornithine, a putative precursor for the biosynthesis of pyrrolysine, is added to the culture medium of transfected HEK293F cells, PCL, not pyrrolysine, is incorporated into hRBP4 at the site of the TAG codon. 6B shows SDS-PAGE, and FIG. 6C shows the purified hRBP4 Phe62PCL (mutant #2) produced in HEK293F cells in the absence (B, lane 1) or presence (B, lane 2) of D-ornithine. Show the mass spectrum. The arrows indicate the full length hRBP4, and the mass obtained was 23166.0 Da, which is close to the expected mass of 23168 Da. Additionally, the mass spectrometry data shown in FIGS. 7-9 show that PCL, but not pyrrolysine, is incorporated into the TAG site at residue 62 of hRBP4 (see Example 2).

1 shows the structure of pyrrolysine (PYL) and a demethylated pyrrolysine analog, pyrroline-carboxy-lysine (PCL) (structure PCL-A or alternative structure PCL-B). The structure of PCL-A (or alternative structure PCL-B) was distinguished from pyrrolysine using high-precision mass spectrometric analysis of the peptide fragment at the site of incorporation (FIGS. 7-9 ). In addition, the structure of PCL was distinguished from pyrrolysine by detection of PCL by mass spectrometry as a free amino acid in the cell lysate (see Fig. 10 and Example 3). In addition, observation of PCL in cell lysates demonstrates that PCL is produced biosynthetically as a free amino acid rather than formed by post-translational modification. 10A and 10C depict the detection of PCL in lysates from HEK293F cells biosynthesized from D-ornithine. 10A is an HPLC trace of lysates from cells transfected with the biosynthetic genes pylB , pylC and pylD and grown in the presence of D-ornithine (bottom trace) and in the absence of D-ornithine (top trace). The lysate chromatogram obtained from cells grown in the presence of D-ornithine is characterized by a peak at 4.13 min elution time (indicated by an asterisk), which in the lysate of the same cells cultured in the absence of D-ornithine does not exist. 10C is the full scan mass spectrum of the HPLC peak at 4.13 min, where the mass obtained (m+1) is consistent with the theoretical mass for PCL. Figure 10B is an HPLC chromatogram showing that lysine is equally abundant in both samples, and the total scanning mass (m+1) spectrum of lysine HPLC at 1.44 min explains the accuracy of the method (Figure 10D).

11 depicts the incorporation of N-ε-cyclopentyloxycarbonyl-L-lysine (CYC) into various hRBP4 TAG mutant proteins in HEK293F cells. 12 shows mass spectrometric verification of CYC incorporation at the TAG site of the hRBP4 mutant Phe62TAG. 13A and 13B (see Example 4) show PCL incorporation as a function of the various precursors listed in the table and shown in FIG. 14A. 13A and 13B also depict direct incorporation of various pyrrolysine analogs (including CYC) into hRBP4 TAG mutant proteins using HEK293F cells. To demonstrate that D-ornithine is a precursor for the biosynthesis of PCL, a potential precursor for PCL (Figure 14A) was identified as pyrrolysine biosynthesis pylB, pylC and pylD genes, pylT/pylS tRNA/pyrrolysyl-tRNA synthetase. It was added to the growth medium of HEK293F cells transfected with paired and hRBP4 TAG mutant DNA. In addition, various pyrrolysine analogs added to the growth medium of HEK293F cells transfected only with the pylT/pylS tRNA/aa-tRNA synthetase pair and hRBP4 TAG mutant DNA are shown in FIG. 14B. Figure 13A is a Western blot of full length hRBP4 protein with anti-His tag antibody, whereas Figure 13B is SDS-PAGE of the same sample after Ni-NTA purification. Although D-arginine (lane 4), which can be metabolized to D-ornithine, exceeds background protein production, as shown, D-ornithine (lane 2) is a better precursor for PCL biosynthesis. Of the pyrrolysine analogs, only CYC produces proteins at levels similar to D-ornithine, while the 3-oxobutanoic acid analog TU3000-016, although measurable, shows very low incorporation. Techniques for the synthesis of various analogs are provided in Example 33. All lanes show low levels of production of full-length proteins, possibly due to low endogenous levels of D-ornithine and metabolites or media components acceptable as PylS substrate.

Incorporation of PCL was evaluated using different combinations of biosynthetic genes pylB, pylC and pylD (Fig. 13C). The data show that only genes pylC and pylD are essential for PCL biosynthesis and subsequent protein incorporation. Most notably, D-proline, other D-proline analogs and D-glutamic acid (Figures 13 and 14) did not show full-length protein production above background. This suggests that the biosynthesis of PCL-A and PCL-B does not follow the pathway suggested in Fig. 2A. However, the incorporation test using 3,4-dihydro-2H-pyrrole-5-carboxylate (P2C) and 1-pyrroline-5-carboxylate (P5C) also produced full-length proteins in Escherichia coli. While failing, both synthetic PCL-A and PCL-B were incorporated with high efficiency (Figure 15A, Example 15). Therefore, the biosynthetic pathway proposed in FIG. 2B may not be a promising pathway.

The incorporation of the precursor (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid (also referred to as Lys-Nε-D-Orn) results in a significant amount of full-length protein. Was obtained (Fig. 15A, Example 15). Furthermore, it was confirmed that the incorporation of (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid only required the presence of the pylS, pylT and PylD genes (Fig. 15B). , Example 15). Together, these observations suggest an alternative route shown in Figs. 16A and 16B. This pathway is followed by coupling of D-ornithine to the epsilon amino group of L-lysine first with the help of PylC, putative D-alanyl-D-alanine ligase, and thus (2S)-2-amino-6-(( It is based on the concept of forming R)-2,5-diaminopentanamido)hexanoic acid. In Fig. 16A, (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid intermediate is D-ornithine: oxygen oxidoreductase (EC 1.4.3.3) or Activated by D-amino-transaminase (EC 2.6.1.21), forming PCL-B by spontaneous cyclization. Alternatively, (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid intermediate in FIG. 16B is D-ornithine:2-oxidoglutarate 5- It is activated by transaminase (EC 2.6.1.13), forming PCL-A by spontaneous cyclization. Both cyclization reactions are similar to those of D-glutamate 5-semialdehyde. One of the activation steps prior to cyclization can putatively be catalyzed by PylD, and the second step requires enzymes for the biosynthesis of PCL. As shown in Fig. 16, it is proposed that methylation of PCL-A by PylB completes the biosynthesis of PYL. Note that in the case of the pathway of Figure 16A, it is likely that this will require the presence of another to-be-discovered Pyl enzyme that forms PCL-A from PCL-B. However, this additional enzyme would not require the alternative pathway shown in 16B. PCL-A formed after activation by PylD and spontaneous cyclization can be directly methylated by PylB to form pyrrolysine. Alternatively, any intermediate or D-ornithine can be a substrate for methylation by PylB if the subsequent enzyme tolerates this modification of the substrate.

It has been found that the pyrrolysyl-tRNA synthetase PylS allows ^{several pyrrolysine analogs to confer tRNA Pyl.} However, these studies also pointed out the importance of the pyrrolysine ring C-5 stereocenter for recognition of the synthetase PylS, where the D-form is required (Polycarpo, CR, Herring, S., Berube, et al. A., Wood, JL, Soll, D., and Ambrogelly, A., (2006), "Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase," FEBS Letters , 580 , 6695-700). As provided herein, consistent with this interpretation, attempts to incorporate a variety of pyrrolysine analogs, including aromatic 5 and 6-membered ring pyrrolysine analogs (Figure 13), show that these aromatic 5 and 6-membered ring analogs Was not an acceptable substrate for PylS (FIG. 14 ). This is possibly due to the lack of a C-5 chiral center, or in certain cases due to a larger sized analog. As noted, N-ε-cyclopentyloxycarbonyl-L-lysine (CYC), a known substrate for PylS, and TU3000-016, a 3-oxobutanoic acid analog, are PylT/PylS tRNA/pyrrolysyl- It was incorporated using a pair of tRNA synthetase (FIG. 14 ). However, analogs similar in size and bulk to N-ε-cyclopentyloxycarbonyl-L-lysine (CYC) were evaluated as not incorporated because possibly lacking the C-5 stereocenter. Thus, PylS appears to be selective depending on chirality at the point of attachment of the pyrroline ring. However, synthetic PCL-B was also incorporated with high efficiency, suggesting that the sp2 achiral carbon at the C-5 position does not favor incorporation in all cases. Currently, the low reactivity of synthetic PCL-B with 2-ABA compared to synthetic PCL-A (Example 44, Figures 56 and 58), and of known enzymes for the conversion of PCL-B to PCL-A in Figure 16A. Absence favors the assignment of pyrrolysine analogs incorporated as PCL-A.

As provided herein, in certain embodiments, when D-ornithine is added to the growth medium as a precursor, biosynthesis of pyrrolysine in mammalian cells (Example 2) and bacterial cells (Examples 8, 9 and 10). It was found that the use of natural genes pylB, pylC and pylD for pyrrolysine produced PCL. In addition, the evaluation of the biosynthesis of PCL using the natural genes pylB and pylC, the natural genes pylB and pylD, or the natural genes pylC and pylD produced PCL only when the pylC and pylD gene products were present. This suggests that when D-ornithine is added to the growth medium as a precursor, the gene product of pylB is not required for the biosynthesis of PCL (see Fig. 13C, Example 4). This was carried out without co-transfection of the gene pylB into mammalian cells, three model proteins, hRBP4 (see Figure 6, Example 2), mIgG1 Fc domain (see Figure 17A, Example 5) and mEPO (Figure 17B, (See Example 6) is supported by the incorporation of PCL. In Escherichia coli, FAS-TE, FGF21 and FKBP are examples of exclusively generating PCL using the natural genes pylB, pylC and pylD (Figs. 18B, 19 and 20, Examples 8, 9 and 10).

The biosynthesis of PCL requires the presence of the biosynthetic genes pylC and pylD, not pylB, in the host cell. In the biosynthesis of pyrrolysine in metanosarcina, it is found that PylD contains the NADH-binding domain of dehydrogenase, thus producing D-1-pyrroline-5-carboxylate from D-proline. Was proposed. However, adding D-proline to the growth medium did not indicate significant PCL incorporation (FIG. 13 ). In addition, 3,4-dihydro-2H-pyrrole-5-carboxylate (herein 1-pyrroline-2-carboxylate; also referred to as P2C) and D-1-pyrroline-5- The addition of carboxylate (P5C) failed to produce the full-length protein, while (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid produced the PCL-containing protein. do. PylC has sequence homology with D-alanyl-D-alanine ligase, and catalyzes the attachment of D-ornithine to the epsilon-amino group of lysine in the biosynthesis of PCL or pyrrolysine (2S)-2-amino- 6-((R)-2,5-diaminopentanamido)hexanoic acid can be provided. Thus, this is not bound to any theory herein, and the biosynthesis of PCL from D-ornithine in mammalian or Escherichia coli cells is the (2S)-2-amino-6- of D-ornithine by PylC. It is assumed that it may be related to the conversion to ((R)-2,5-diaminopentanamido)hexanoic acid (Figure 16B). As shown in Figure 16B, PylD is (2S)-2-amino-6-((R)-2,5-diaminopentanamido) hexanoic acid spontaneously cyclizes to PCL-A ((S )-2-amino-6-((R)-2-amino-5-oxopentanamido)hexanoic acid). Alternatively, PCL-B will form spontaneously if PylD has a D-amino acid transaminase or D-ornithine:oxygen oxidoreductase-like activity (Figure 16A).

In another embodiment, expression of mouse EGF and mouse TNF-α in Escherichia coli (Figure 21 and Examples 13 and 14) results in a protein mixture with PCL or pyrrolysine incorporated at the TAG site. The incorporation of pyrrolysine depended on the presence of the pylB gene, but a homogeneous preparation of PCL-containing protein was observed in the absence of pylB (Fig. 21 and Examples 11 and 12). Thus, it experimentally demonstrates PylB as a methyltransferase in the biosynthesis of pyrrolysine. Even in the presence of the pylB gene, proteins containing relative amounts of PCL and pyrrolysine typically differ from fermentation with the more promising PCL protein. These observations suggest that methyltransferase activity or methyl-donating substrates or necessary cofactors are limited for efficient pyrrolysine biosynthesis in Escherichia coli and mammalian cells.

In addition, the gene product of pylB may not be expressed efficiently. Thus, in certain embodiments, the modified pylB gene is used for biosynthesis of pyrrolysine or other pyrrolysine analogs. Accordingly, provided herein are methods for the biosynthesis of pyrrolysine, PCL and other pyrrolysine analogs in Escherichia coli, mammals and other host cells, wherein one or more of the pylB, pylC and pylD genes have been modified. Such modifications may include the use of homologous genes from other organisms, such as, but not limited to, other species of metanosarcinae or mutated genes. In certain embodiments, site-directed mutagenesis is used, while in other embodiments random mutagenesis in combination with selection is used. This method also includes adding the DNA of the desired protein to incorporate pyrrolysine, PCL or a pyrrolysine analog into the protein, and including the pylT and pylS genes. In certain embodiments, the modified pylB gene, the native pylC, pylD, pylT and pylS genes are used to biosynthetize and incorporate pyrrolysine. In other embodiments, pyrrolysine analogs other than PCL are biosynthetically generated and incorporated using the modified pylB and pylC genes, the native pylD, pylT and pylS genes. In other embodiments, pyrrolysine analogs other than PCL are biosynthetically generated and incorporated using the modified pylB and pylD genes, the native pylC, pylT and pylS genes. In other embodiments, the pylT, pylS, pylB, pylC and pylD genes are optionally modified to improve incorporation of pyrrolysine, PCL or other pyrrolysine analogs into the protein.

Furthermore, for certain embodiments, the biosynthesis of pyrrolysine, PCL and/or other pyrrolysine analogs from D-ornithine or the formation of intermediates in the biosynthesis of pyrrolysine may be limited by the function of host enzymes and proteins. . In certain embodiments, the low activity or concentration of one or more host enzymes may limit the formation of intermediates required for the biosynthesis of pyrrolysine, PCL or other pyrrolysine analogs. In certain embodiments, the activity of the host enzyme can convert intermediates from pathways leading to pyrrolysine, PCL or other pyrrolysine analogs to other metabolic pathways, or can limit the formation of such intermediates. Accordingly, provided herein are methods for the biosynthesis of pyrrolysine, PCL and other pyrrolysine analogs in Escherichia coli, mammals or other host cells, in which one or more host enzymes have been modified. These methods include overexpression, activation, inhibition or inhibition of such host enzymes by genetic or chemical means, addition of DNA encoding these host enzymes, addition of silencing RNA (siRNA) to inhibit mRNA translation, and D- The addition of cofactors necessary for the formation of the intermediate from ornithine is included, but is not limited thereto.

Site-specific incorporation of biosynthetically generated PCL at the TAG-encoded site was also carried out at four sites of the Fc domain of mouse IgG1 (see Example 5 and Figure 17A) and at 11 sites of erythropoietin (EPO) ( See Example 6 and Fig. 17B) was achieved. PCL incorporation for both sets of proteins was achieved using D-ornithine added to the medium as a precursor in HEK293F mammalian cells infected with the native genes pylT, pylS, pylC and pylD. Figure 17A is an SDS-PAGE showing four full-length (FL) mFc proteins, where PCL is HEK293F cells and D co-transfected with respective TAG mutant constructs of mFc, pCMVpylT, pCMVpylS, pCMVpylC and pCMVpylD (Figure 4A). -Cells grown in the presence of ornithine were incorporated into four sites of the mouse IgG1 Fc domain. Figure 17B is an SDS-PAGE showing 11 full-length (FL) mEPO proteins, where PCL is in the presence of HEK293F cells and D-ornithine co-transfected with TAG mutant constructs of mEPO, pCMVpylT, pCMVpylS, pCMVpylC and pCMVpylD. The grown cells were incorporated into 11 sites of mouse EPO.

Site-specific incorporation of biosynthetically generated PCL at the TAG-encoded site was also achieved using Escherichia coli cells (see Example 7). A plasmid encoding pylB, pylC, pylD, pylS and pylT, pARA-pylSTBCD, was constructed (Fig. 6A). Two sites of the thioesterase domain of human fatty acid synthetase (FAS-TE) (see Examples 8 and 18), one site of FKBP-12 (see Examples 9 and 19) and fibroblast growth factor Incorporation of PCL-A (or PCL-B) at 20 sites (see Example 10 and FIG. 20) of 21 (FGF21) results in Escherichia coli cells with a second expression of pARA-pylSTBCD and a gene for the protein of interest. Transformed with plasmid and achieved by adding D-ornithine to the growth medium during protein expression.

18 shows SDS-PAGE and mass spectra for PCL incorporation at two sites of thioesterase of human fatty acid synthetase (FAS-TE). FAS-TE Tyr2454PCL was expressed and both soluble and insoluble protein fractions were purified by Ni-NTA. FAS-TE Leu2222PCL/Leu2223Ile was expressed in clone culture with or without D-ornithine. Ni-NTA elution is shown on the gel. The mass obtained for each is consistent with what is expected. Figure 19 shows SDS-PAGE and mass spectrum for PCL incorporation at one site of FKBP-12. The mass obtained (12085.6 Da) is consistent with that expected for single site incorporation of PCL (12084 Da). Further, the determination of FKBP12-Ile90PCL is shown in Fig. 19. 20 depicts SDS-PAGE showing the incorporation of PCL into FGF21 at multiple sites.

The incorporation of pyrrolysine (PYL) and PCL was found to depend on the presence of the pylB gene in the expression system. 21A shows SDS-PAGE analysis of Pyl- or PCL-incorporation into mTNF-α by the codon (CAA) of Gln21 mutated to the TAG stop codon (Example 13). When D-ornithine is present, lanes 2 and 4 show similar protein expression levels with and without pylB, respectively. Lanes 3 (with pylB) and 5 (without pylB) show that the protein is not expressed in the absence of D-ornithine.

In addition, mEGF Tyr10TAG was expressed in the presence of pylB, pylC, pylD, pylT and pylS genes in Escherichia coli using D-ornithine as a precursor (Example 14). The circular MS spectrum (Fig. 21C, bottom) suggested a mixture of proteins in which PYL and PCL were incorporated. The MS peak of the Pyl-containing mEGF at 7309 Da is approximately 6 times as large as the peak of the PCL-containing mEGF at 7295 Da. The incorporation of PYL and PCL at the TAG position was verified by tandem MS spectra of the N-terminal peptides MNSYPGCPSS(PCL )DGYCLNGGVCM (SEQ ID NO:32) and MNSYPGCPSS( Pyl )DGYCLNGGVCM (SEQ ID NO:33) of the mEGF Tyr10TAG mutant protein. Quantification from the relative precursor mass abundance of the different peptides showed that PYL was 5-10 times more abundant than PCL, which is roughly consistent with the circular mass measurements (Figure 21C, bottom). When mEGF Tyr10TAG was expressed in the presence of pylB, the circular MS spectrum suggested only PCL incorporation (Fig. 21C, top).

Similarly, when mTNF-α Gln21TAG was expressed using D-ornithine in the presence of pylB, pylC, pylD, pylT and pylS genes in Escherichia coli, tandem MS showed the peptide NH( Pyl )VEEQLEWLSQR ( SEQ ID NO: 34) and NH ( PCL ) VEEQLEWLSQR (SEQ ID NO: 35) incorporation of PYL and PCL was verified. In contrast to mEGF Tyr10TAG, quantification from the relative precursor mass abundance of the different peptides identified by tandem MS showed that in the mTNF-α Gln21TAG protein, PCL was 7 times more abundant than PYL. mTNF-α Gln21TAG was expressed in the absence of the pylB gene, and quantification from the relative precursor mass abundance of different peptides in tandem MS measurements reveals only the PCL protein within the dynamic range of the experiment. These experiments indicate that PYL incorporation is strictly dependent on the presence of pylB, a putative methyltransferase, in the biosynthesis of Pyl. However, the relative ratio of PCL and PYL incorporated into a protein in the presence of the pylB gene appears to vary from protein to protein and from fermentation experiments, and thus may depend on the type of expression vector, growth conditions and/or other characteristics of the host cell culture. .

Provided herein are amino acids having the structures of Formula V and Formula VI,

<Formula V>

<Formula VI>

Here, the compound of Formula V or Formula VI is biosynthetically produced in cells containing the pylB gene, the pylC gene, and the pylD gene, and the cells are in contact with the growth medium containing the precursor. Meanwhile, in another embodiment, the compound of Formula V or Formula VI is biosynthetically produced in cells containing the pylC gene and the pylD gene, and the cells are in contact with the growth medium containing the precursor. In certain embodiments of this biosynthesis, the precursor is ornithine or arginine. In certain embodiments of this biosynthesis, the precursor is D-ornithine or D-arginine. In certain embodiments of this biosynthesis, the precursor is (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid. In another embodiment of this biosynthesis, the precursor is (2S)-2-amino-6-(2,5-diaminopentanamido)hexanoic acid.

As also provided herein, the cells in which the compounds of formulas V and VI are biosynthesized further comprise a pylS gene and a pylT gene, and the compounds of formula V or VI are aminoacyl tRNA synthetase and one or more selectors of mRNA in cells. It is incorporated into the protein within the cell by the tRNA that recognizes the codon. This aminoacyl tRNA synthetase is a gene product of the pylS gene, and tRNA is a gene product of the pylT gene. In certain embodiments, the compound of Formula V or Formula VI is incorporated into an intracellular protein by an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS), wherein O-RS is Formula V or The O-tRNA was aminoacylated by the compound of Formula VI, and the O-tRNA recognized one or more selector codons of the mRNA in the cell.

The selector codon for the incorporation of pyrrolysine, PCL and/or other pyrrolysine analogs such as compounds of formula V and formula VI is the amber codon (TAG).

Cells used for biosynthesis and/or incorporation of pyrrolysine, PCL and/or other pyrrolysine analogs, such as compounds of formula V and formula VI, are prokaryotic or eukaryotic cells. In certain embodiments, prokaryotic cells include, but are not limited to, Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis, and Bacillus subtilis cells. In other embodiments eukaryotic cells include, but are not limited to, mammalian cells, yeast cells, fungal cells, plant cells, or insect cells. These mammalian cells include human embryonic kidney (HEK293F) cells, human epithelial carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-21) cells, and Chinese hamster ovary. (CHO) cells are included, but are not limited thereto. In certain embodiments, yeast cells include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells include Spodoptera frugiperda sf9 and sf21 cells, Trichoflucia ni (BTI TN-5B1-4 or High-Five™) cells, and Mammestra brassicae cells, It is not limited.

In another aspect provided herein, pyrrolysine, PCL and/or other pyrrolysine analogs, such as compounds of Formula V and Formula VI, are biosynthesized in a feeder cell in contact with a growth medium comprising a precursor, and the feeder cell is It contains the pylB gene, the pylC gene and the pylD gene. In other embodiments, pyrrolysine, PCL and/or pyrrolysine analogs, such as compounds of Formula V and Formula VI, are biosynthesized in a feeder cell in contact with a growth medium comprising a precursor, and the feeder cell is the pylC gene and pylD Contains genes. These biosynthesized pyrrolysine, PCL and/or other pyrrolysine analogs, such as compounds of formula V and formula VI, are secreted from feeder cells into the growth medium, whereby they are taken up by the second cell and subsequently second It is incorporated into the protein synthesized in the cell. This second cell contains the pylS gene and the pylT gene. Pyrrolysine, PCL and/or other pyrrolysine analogs, such as compounds of Formula V and Formula VI, are in a second cell by means of an aminoacyl tRNA synthetase and a tRNA that recognizes one or more selector codons of the mRNA in the second cell. It is incorporated into the protein. This aminoacyl tRNA synthetase is a gene product of the pylS gene, and tRNA is a gene product of the pylT gene. In certain embodiments, the compound of Formula V or Formula VI is incorporated into a second intracellular protein by an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS), wherein O-RS is the formula O-tRNA was aminoacylated by a compound of V or Formula VI, and the O-tRNA recognized one or more selector codons of the mRNA in the second cell.

In certain embodiments of such biosynthesis using feeder cells, the precursor is ornithine or arginine. In certain embodiments of this biosynthesis, the precursor is D-ornithine or D-arginine. In certain embodiments, the precursor is (2S)-2-amino-6-(2,5-diaminopentanamido)hexanoic acid. In certain embodiments, the precursor is (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid.

The selector codon for the incorporation of pyrrolysine, PCL and/or other pyrrolysine analogs, such as compounds of formula V and formula VI biosynthesized using feeder cells, is the amber codon (TAG).

In certain embodiments, the second cells are of the same type as the feeder cells, while in other embodiments, the second cells are of a different cell type than the feeder cells. The cells used for this biosynthesis and/or incorporation of pyrrolysine, PCL and/or other pyrrolysine analogs such as compounds of formula V and formula VI are prokaryotic or eukaryotic cells. In certain embodiments, prokaryotic cells include, but are not limited to, Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis, and Bacillus subtilis cells. In other embodiments eukaryotic cells include, but are not limited to, mammalian cells, yeast cells, fungal cells, plant cells, or insect cells. These mammalian cells include human embryonic kidney (HEK293F) cells, human epithelial carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-21) cells, and Chinese hamster ovary. (CHO) cells are included, but are not limited thereto. In certain embodiments, yeast cells include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells include Spodoptera frugiperda sf9 and sf21 cells, Trichoflucia ni (BTI TN-5B1-4 or High-Five™) cells, and Mammestra brassicae cells, It is not limited.

In another aspect provided herein, pyrrolysine, PCL and/or other pyrrolysine analogs, such as compounds of Formula V and Formula VI, are biosynthesized in feeder cells, and then such biosynthesized pyrrolysine and/or PCL, such as The compounds of formula (V) and (VI) are purified from the feeder cell culture and added to the growth medium of the second culture containing the second cells. These purified pyrrolysine, PCL and/or other pyrrolysine analogs are then incorporated into the synthesized protein in the second cell. In certain embodiments, pyrrolysine, PCL and/or other pyrrolysine analogs, such as compounds of Formula V and Formula VI, are biosynthesized in a feeder cell in contact with a growth medium comprising a precursor, and the feeder cell is a pylB gene, It contains the pylC gene and the pylD gene. In other embodiments, pyrrolysine, PCL and/or other pyrrolysine analogs, such as compounds of Formula V and Formula VI, are biosynthesized in a feeder cell in contact with a growth medium comprising a precursor, and the feeder cell comprises a pylC gene and It contains the pylD gene. The second cell used in this aspect contains the pylS gene and the pylT gene. Pyrrolysine, PCL and/or other pyrrolysine analogs, such as compounds of Formula V and Formula VI, are in a second cell by means of an aminoacyl tRNA synthetase and a tRNA that recognizes one or more selector codons of the mRNA in the second cell. It is incorporated into the protein. This aminoacyl tRNA synthetase is a gene product of the pylS gene, and tRNA is a gene product of the pylT gene. In certain embodiments, the compound of Formula V or Formula VI is incorporated into a second intracellular protein by an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS), wherein O-RS is the formula O-tRNA was aminoacylated by a compound of V or Formula VI, and the O-tRNA recognized one or more selector codons of the mRNA in the second cell.

In certain embodiments of such biosynthesis using feeder cells, the precursor is ornithine or arginine. In certain embodiments of this biosynthesis, the precursor is D-ornithine or D-arginine. In certain embodiments, the precursor is (2S)-2-amino-6-(2,5-diaminopentanamido)hexanoic acid. In certain embodiments, the precursor is (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid.

The selector codon for the incorporation of pyrrolysine, PCL and/or other pyrrolysine analogs, such as compounds of formula V and formula VI biosynthesized using feeder cells, is the amber codon (TAG).

In certain embodiments, the second cells are of the same type as the feeder cells, while in other embodiments, the second cells are of a different cell type than the feeder cells. The cells used for this biosynthesis and/or incorporation of pyrrolysine, PCL and/or other pyrrolysine analogs such as compounds of formula V and formula VI are prokaryotic or eukaryotic cells. In certain embodiments, prokaryotic cells include, but are not limited to, Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis, and Bacillus subtilis cells. In other embodiments eukaryotic cells include, but are not limited to, mammalian cells, yeast cells, fungal cells, plant cells, or insect cells. These mammalian cells include human embryonic kidney (HEK293F) cells, human epithelial carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-21) cells, and Chinese hamster ovary. (CHO) cells are included, but are not limited thereto. In certain embodiments, yeast cells include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells include Spodoptera frugiperda sf9 and sf21 cells, Trichoflucia ni (BTI TN-5B1-4 or High-Five™) cells, and Mammestra brassicae cells, It is not limited.

In addition, an amino acid having the structure of the following formula (VII) is provided herein,

<Formula VII>

Here, the compound of Formula VII, or an isomer or tautomer thereof, is biosynthetically produced in cells containing the pylB gene, the pylC gene and the pylD gene, and the cell is D-ornithine or D-arginine or (2S)-2 -Amino-6-((R)-2,5-diaminopentanamido)hexanoic acid or 2,5-diamino-3-methylpentanoic acid or (2S)-2-amino-6-(2,5 It is in contact with a growth medium containing -diaminopentanamido)hexanoic acid. Meanwhile, in another embodiment, such a compound of Formula VII is produced biosynthetically in cells containing the pylC gene and the pylD gene, and the cells are contacted with a growth medium containing 2,5-diamino-3-methylpentanoic acid. I'm doing it. In certain embodiments, the cells are contacted with a growth medium containing D-2,5-diamino-3-methylpentanoic acid. In certain embodiments, the 2,5-diamino-3-methylpentanoic acid is (2R,3S)-2,5-diamino-3-methylpentanoic acid. In certain embodiments, the 2,5-diamino-3-methylpentanoic acid is (2R,3R)-2,5-diamino-3-methylpentanoic acid.

Cells in which the compound of Formula VII is biosynthetically produced further contains the pylS gene and the pylT gene, and the compound of Formula VII is intracellular by aminoacyl tRNA synthetase and tRNA that recognizes one or more selector codons of mRNA in the cell. Is incorporated into the protein. This aminoacyl tRNA synthetase is a gene product of the pylS gene, and tRNA is a gene product of the pylT gene. In certain embodiments, the compound of formula VII is incorporated into an intracellular protein by an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS), wherein the O-RS is by the compound of formula VII. The O-tRNA was aminoacylated, and the O-tRNA recognized one or more selector codons of the mRNA in the cell.

The selector codon for incorporation of the compound of formula VII is the amber codon (TAG).

Cells used for biosynthesis and/or incorporation of the compounds of formula VII are prokaryotic or eukaryotic cells. In certain embodiments, prokaryotic cells include, but are not limited to, Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis, and Bacillus subtilis cells. In other embodiments eukaryotic cells include, but are not limited to, mammalian cells, yeast cells, fungal cells, plant cells, or insect cells. These mammalian cells include human embryonic kidney (HEK293F) cells, human epithelial carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-21) cells, and Chinese hamster ovary. (CHO) cells are included, but are not limited thereto. In certain embodiments, yeast cells include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells include Spodoptera frugiperda sf9 and sf21 cells, Trichoflucia ni (BTI TN-5B1-4 or High-Five™) cells, and Mammestra brassicae cells, It is not limited.

In certain embodiments, one or more pyrrolysine, PCL and/or other pyrrolysine analogs are incorporated into proteins, polypeptides and/or peptides using the methods provided herein, and such pyrrolysines and/or PCLs are incorporated herein. It is derivatized using the method provided in.

PCL And of pyrrolysine Derivatization

Provided herein are proteins, polypeptides or peptides having a structure according to Formula I below.

<Formula I>

In the above formula,

R ₁ is H or an amino terminal modified group;

R ₂ is OH or a carboxy terminal modified group;

n is an integer from 1 to 5000;

Each AA is independently selected from an amino acid residue, a residue having the structure of formula A-1, and a residue having the structure of formula B-1;

, 여기서

, here

R ₆ is H or C ₁ alkyl, and at least one AA is a pyrrolysine or pyrrolysine analog having the structure of formula A-1 or B-1, or an isomer thereof.

In certain embodiments of these compounds of formula I, R ₆ is H, and the moiety having the structure of formula A-1 and the moiety having the structure of formula B-1 are each of the following structures A-2 and B-2: Is a moiety having, or an isomer thereof.

Accordingly, proteins, polypeptides or peptides having a structure according to formula (I) below are also provided herein.

<Formula I>

In the above formula,

R ₁ is H or an amino terminal modified group;

R ₂ is OH or a carboxy terminal modified group;

n is an integer from 1 to 5000;

Each AA is independently selected from an amino acid residue, a residue having the structure of formula A-2, and a residue having the structure of formula B-2;

At least one AA is a pyrrolysine or pyrrolysine analog having the structure of Formula A-2 or Formula B-2, or an isomer thereof.

In certain embodiments, n is an integer from 1 to 4000. In certain embodiments n is an integer from 1 to 3000. In certain embodiments n is an integer from 1 to 2000. In certain embodiments n is an integer from 1 to 1000. In certain embodiments n is an integer from 1 to 700. In certain embodiments n is an integer from 1 to 800. In certain embodiments n is an integer from 1 to 600. In certain embodiments n is an integer from 1 to 500. In certain embodiments n is an integer from 1 to 400. In certain embodiments n is an integer from 1 to 300. In certain embodiments n is an integer from 1 to 200. In certain embodiments n is an integer from 1 to 100. In certain embodiments n is an integer from 1 to 90. In certain embodiments n is an integer from 1 to 80. In certain embodiments n is an integer from 1 to 70. In certain embodiments n is an integer from 1 to 60. In certain embodiments n is an integer from 1 to 50. In certain embodiments n is an integer from 1 to 40. In certain embodiments n is an integer from 1 to 30. In certain embodiments n is an integer from 1 to 20. In certain embodiments n is an integer from 1 to 10. In certain embodiments n is an integer from 1 to 5.

In addition, proteins, polypeptides and/or proteins of formula I comprising mixing a protein, polypeptide and/or peptide of formula I containing at least one pyrrolysine and/or PCL moiety with a reagent having the structure of formula III Alternatively, methods for site-specific labeling of peptides are provided herein.

<Formula III>

In the above formula,

R _3, R ₅ and each R ₄ is H, -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX ¹ are independently selected;

L is a bond, C _{1 - 8} alkylene, halo-substituted -C _{1 - 8} alkylene, hydroxy-substituted -C _{1 - 8} alkylene, C _2-8 alkenylene group, a halo-substituted -C _{2 - 8} alkenylene, hydroxy-substituted ₂ -C ₈ alkenylene, polyalkylene glycols, poly (ethylene ^{glycol), -O (CR 11 R 12} ) k -, -S (CR 11 R 12) k - , -S(O) _k (CR ¹¹ R ¹² ) _k -, -O(CR ¹¹ R ¹² ) _k -NR ¹¹ C(O)-, -O(CR ¹¹ R ¹² ) _k C(O)NR ^11- , -C(O)-, -C(O)(CR ¹¹ R ¹² ) _k -, -C(S)-, -C(S)(CR ¹¹ R ¹² ) _k -, -C(O)NR ¹¹ -, -NR ¹¹ C(O)-, -NR ¹¹ (CR ¹¹ R ¹² ) _k -, -CONR ¹¹ (CR ¹¹ R ¹² ) _k -, -N(R ¹¹ )CO(CR ¹¹ R ¹² ) _k- , -C(O)NR ¹¹ (CR ¹¹ R ¹² ) _k -, -NR ¹¹ C(O)(CR ¹¹ R ¹² ) _k -, wherein each R ¹¹ and R ¹² is independently H, C _{1 - 8} alkyl, halo-substituted -C _1-8 alkyl, or hydroxy-substituted -C _{1 - 8} is alkyl, k is an integer from 1 to 12;

X ¹ is a label, dye, polymer, water-soluble polymer, polyalkylene glycol, poly(ethylene glycol), derivative of poly(ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Label, reactive compound; Resin, peptide, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelating agent, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, PCR probe, antisense polynucleotide, ribo-oligonucleotide, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labeling , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocausing moieties, actinic radiation excitable moieties, ligands, photoisomerizing moieties, biotin, biotin analogs , Heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-activators, amino thio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups , Chromophore group, chemiluminescent group, fluorescent moiety, electron high density group, magnetic group, intercalating group, chelating group, chromophore, energy transfer agent, biologically active agent, detectable label, small molecule, inhibitory ribonucleic acid, siRNA, radionucleotide , Neutron-capturing agent, biotin derivative, quantum dot(s), nanotransmitter, radiotransmitter, abzyme, enzyme, activated complex activator, virus, toxin, adjuvant, TLR2 agonist, TLR4 agonist, TLR7 Agonist, TLR9 agonist, TLR8 agonist, T-cell epitope, phospholipid, LPS-like molecule, keyhole limpet hemocyanin (KLH), immunogenic hapten, aglycan, allergen, angiostatin, antihormonal, antioxidant, Aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, mimotopes, receptors, reverse micelles, detergents, immune response enhancers, fluorescent dyes, FRET reagents, radiation-imaging probes, other spectroscopic probes, prodrugs, for immunotherapy Toxin, solid support, -CH ₂ CH ₂ -(OCH ₂ CH ₂ O) _p -OX ² ,- _{O- (CH 2 CH 2 O)} p CH 2 CH 2 -X 2 , and any combination of these (where p is 1 to 10,000, X ² is H, C _{1 - 8} alkyl, protecting group or a terminal functional group being ) Is selected from.

In addition, formula I, comprising mixing and reacting under appropriate conditions a protein, polypeptide and/or peptide of formula I containing at least one pyrrolysine and/or PCL moiety with a reagent having the structure of formula IV Provided herein are methods for site-specific labeling of proteins, polypeptides and/or peptides of.

<Formula IV>

In the above formula,

R ₅ is -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, aryl, heteroaryl, heterocycloalkyl or Selected from cycloalkyl, and -LX ^1;

A is C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ heterocycloalkyl, 5-6 membered monocyclic aryl, 5-6 membered monocyclic heteroaryl, 9-10 membered fused bicyclic ring or 13- a 14-membered fused tricyclic ring, where A is -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, Optionally substituted with 1 to 5 substituents independently selected from aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX ^1;

L is a bond, C _{1 - 8} alkylene, halo-substituted -C _{1 - 8} alkylene, hydroxy-substituted -C _{1 - 8} alkylene, C _2-8 alkenylene group, a halo-substituted -C _{2 - 8} alkenylene, hydroxy-substituted ₂ -C ₈ alkenylene, polyalkylene glycols, poly (ethylene ^{glycol), -O (CR 11 R 12} ) k -, -S (CR 11 R 12) k - , -S(O) _k (CR ¹¹ R ¹² ) _k -, -O(CR ¹¹ R ¹² ) _k -NR ¹¹ C(O)-, -O(CR ¹¹ R ¹² ) _k C(O)NR ^11- , -C(O)-, -C(O)(CR ¹¹ R ¹² ) _k -, -C(S)-, -C(S)(CR ¹¹ R ¹² ) _k -, -C(O)NR ¹¹ -, -NR ¹¹ C(O)-, -NR ¹¹ (CR ¹¹ R ¹² ) _k -, -CONR ¹¹ (CR ¹¹ R ¹² ) _k -, -N(R ¹¹ )CO(CR ¹¹ R ¹² ) _k- , -C(O)NR ¹¹ (CR ¹¹ R ¹² ) _k -, -NR ¹¹ C(O)(CR ¹¹ R ¹² ) _k -, wherein each R ¹¹ and R ¹² is independently H, C _{1 - 8} alkyl, halo-substituted -C _1-8 alkyl, or hydroxy-substituted -C _{1 - 8} is alkyl, k is an integer from 1 to 12;

X ¹ is a label, dye, polymer, water-soluble polymer, polyalkylene glycol, poly(ethylene glycol), derivative of poly(ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Label, reactive compound; Resin, peptide, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelating agent, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, PCR probe, antisense polynucleotide, ribo-oligonucleotide, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labeling , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocausing moieties, actinic radiation excitable moieties, ligands, photoisomerizing moieties, biotin, biotin analogs , Heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-activators, amino thio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups , Chromophore group, chemiluminescent group, fluorescent moiety, electron high density group, magnetic group, intercalating group, chelating group, chromophore, energy transfer agent, biologically active agent, detectable label, small molecule, inhibitory ribonucleic acid, siRNA, radionucleotide , Neutron-capturing agent, biotin derivative, quantum dot(s), nanotransmitter, radiotransmitter, abzyme, enzyme, activated complex activator, virus, toxin, adjuvant, TLR2 agonist, TLR4 agonist, TLR7 Agonist, TLR9 agonist, TLR8 agonist, T-cell epitope, phospholipid, LPS-like molecule, keyhole limpet hemocyanin (KLH), immunogenic hapten, aglycan, allergen, angiostatin, antihormonal, antioxidant, Aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, mimotopes, receptors, reverse micelles, detergents, immune response enhancers, fluorescent dyes, FRET reagents, radiation-imaging probes, other spectroscopic probes, prodrugs, for immunotherapy Toxin, solid support, -CH ₂ CH ₂ -(OCH ₂ CH ₂ O) _p -OX ² ,- _{O- (CH 2 CH 2 O)} p CH 2 CH 2 -X 2 , and any combination of these (where p is 1 to 10,000, X ² is H, C _{1 - 8} alkyl, protecting group or a terminal functional group being ) Is selected from.

In certain embodiments k is an integer from 1 to 11. In certain embodiments k is an integer from 1 to 10. In certain embodiments k is an integer from 1 to 9. In certain embodiments k is an integer from 1 to 8. In certain embodiments k is an integer from 1 to 7. In certain embodiments k is an integer from 1 to 6. In certain embodiments k is an integer from 1 to 5. In certain embodiments k is an integer from 1 to 4. In certain embodiments k is an integer from 1 to 3. In certain embodiments k is an integer from 1 to 2.

In certain embodiments p is an integer from 1 to 8000. In certain embodiments p is an integer from 1 to 7000. In certain embodiments p is an integer from 1 to 6000. In certain embodiments p is an integer from 1 to 5000. In certain embodiments p is an integer from 1 to 4000. In certain embodiments p is an integer from 1 to 3000. In certain embodiments p is an integer from 1 to 2000. In certain embodiments p is an integer from 1 to 1000. In certain embodiments p is an integer from 1 to 500. In certain embodiments p is an integer from 1 to 400. In certain embodiments p is an integer from 1 to 300. In certain embodiments p is an integer from 1 to 200. In certain embodiments p is an integer from 1 to 100. In certain embodiments p is an integer from 1 to 90. In certain embodiments p is an integer from 1 to 80. In certain embodiments p is an integer from 1 to 70. In certain embodiments p is an integer from 1 to 60. In certain embodiments p is an integer from 1 to 50. In certain embodiments p is an integer from 1 to 40. In certain embodiments p is an integer from 1 to 30. In certain embodiments p is an integer from 1 to 20. In certain embodiments p is an integer from 1 to 10. In certain embodiments p is an integer from 1 to 5.

Non-limiting examples of compounds of formula IV include:

이고, PADRE는

이고, BG1은

이고, BG2는

이고, 여기서 *는 포스포티오에이트 연결부를 나타낸다.In the above formula, the compound having at least one polyethylene glycol (PEG) moiety has an average molecular weight in the range of 1000 Da to 50 kDa, where n is 20 to 1200, and exPADRE is

And PADRE is

And BG1 is

And BG2 is

And, where * represents a phosphorothioate linking moiety.

In certain embodiments, proteins, polypeptides and/or peptides containing at least one pyrrolysine and/or PCL derivatized using the methods provided herein have the structure of Formula II:

<Formula II>

In the above formula,

R ₁ is H or an amino terminal modified group;

R ₂ is OH or a carboxy terminal modified group;

n is an integer from 1 to 5000;

Each BB is an amino acid residue, a pyrrolysine analog amino acid residue having a structure of the following formula A-2, a pyrrolysine analog amino acid residue having a structure of the following formula B-2, and a pyrrolyse having a structure of the following formula C-1. A new analog amino acid residue, a pyrrolysine analog amino acid residue having a structure of the following formula D-1, a pyrrolysine analog amino acid residue having a structure of the following formula E-1, a pyrrolysine analog having a structure of the following formula F-1 Amino acid residue, a pyrrolysine analog amino acid residue having the structure of the following formula G-1, a pyrrolysine analog amino acid residue having the structure of the following formula H-1, a pyrrolysine analog amino acid residue having the structure of the following formula I-1 , A pyrrolysine analog amino acid residue having a structure of Formula J-1, a pyrrolysine analog amino acid residue having a structure of Formula K-1, and a pyrrolysine analog amino acid residue having a structure of Formula L-1, or Independently selected from its isomers;

, 여기서

, here

R _3, R ₅ and each R ₄ is H, -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX ¹ are independently selected;

R ₆ is H or C ₁ alkyl;

A is C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ heterocycloalkyl, 5-6 membered monocyclic aryl, 5-6 membered monocyclic heteroaryl, 9-10 membered fused bicyclic ring or 13- a 14-membered fused tricyclic ring, where A is -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, Optionally substituted with 1 to 5 substituents independently selected from aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX ^1;

L is a bond, C _{1 - 8} alkylene, halo-substituted -C _{1 - 8} alkylene, hydroxy-substituted -C _{1 - 8} alkylene, C _2-8 alkenylene group, a halo-substituted -C _{2 - 8} alkenylene, hydroxy-substituted ₂ -C ₈ alkenylene, polyalkylene glycols, poly (ethylene ^{glycol), -O (CR 11 R 12} ) k -, -S (CR 11 R 12) k - , -S(O) _k (CR ¹¹ R ¹² ) _k -, -O(CR ¹¹ R ¹² ) _k -NR ¹¹ C(O)-, -O(CR ¹¹ R ¹² ) _k C(O)NR ^11- , -C(O)-, -C(O)(CR ¹¹ R ¹² ) _k -, -C(S)-, -C(S)(CR ¹¹ R ¹² ) _k -, -C(O)NR ¹¹ -, -NR ¹¹ C(O)-, -NR ¹¹ (CR ¹¹ R ¹² ) _k -, -CONR ¹¹ (CR ¹¹ R ¹² ) _k -, -N(R ¹¹ )CO(CR ¹¹ R ¹² ) _k- , -C(O)NR ¹¹ (CR ¹¹ R ¹² ) _k -, -NR ¹¹ C(O)(CR ¹¹ R ¹² ) _k -, wherein each R ¹¹ and R ¹² is independently H, C ₁ and _8, and the alkyl, k is an integer from 1 to 12, _{- 8} alkyl, halo-substituted -C _{1 - 8} alkyl or hydroxy-substituted -C ₁

X ¹ is a label, dye, polymer, water-soluble polymer, polyalkylene glycol, poly(ethylene glycol), derivative of poly(ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Label, reactive compound; Resin, peptide, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelating agent, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, PCR probe, antisense polynucleotide, ribo-oligonucleotide, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labeling , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocausing moieties, actinic radiation excitable moieties, ligands, photoisomerizing moieties, biotin, biotin analogs , Heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-activators, amino thio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups , Chromophore group, chemiluminescent group, fluorescent moiety, electron high density group, magnetic group, intercalating group, chelating group, chromophore, energy transfer agent, biologically active agent, detectable label, small molecule, inhibitory ribonucleic acid, siRNA, radionucleotide , Neutron-capturing agent, biotin derivative, quantum dot(s), nanotransmitter, radiotransmitter, abzyme, enzyme, activated complex activator, virus, toxin, adjuvant, TLR2 agonist, TLR4 agonist, TLR7 Agonist, TLR9 agonist, TLR8 agonist, T-cell epitope, phospholipid, LPS-like molecule, keyhole limpet hemocyanin (KLH), immunogenic hapten, aglycan, allergen, angiostatin, antihormonal, antioxidant, Aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, mimotopes, receptors, reverse micelles, detergents, immune response enhancers, fluorescent dyes, FRET reagents, radiation-imaging probes, other spectroscopic probes, prodrugs, for immunotherapy Toxin, solid support, -CH ₂ CH ₂ -(OCH ₂ CH ₂ O) _p -OX ² ,- _{O- (CH 2 CH 2 O)} p CH 2 CH 2 -X 2 , and any combination of these (where p is 1 to 10,000, X ² is H, C _{1 - 8} alkyl, protecting group or a terminal functional group being ) Is selected from,

At least one AA is a pyrrolysine analog amino acid residue having the structure of Formula A-2 or Formula B-2, or at least one BB is Formula C-1 or Formula D-1 or Formula E-1 or Formula F-1 Or a pyrrolysine analog amino acid residue having the structure of Formula G-1 or Formula H-1 or Formula I-1 or Formula J-1 or Formula K-1 or Formula L-1, or an isomer thereof.

In certain embodiments, k is an integer from 1 to 11. In certain embodiments, k is an integer from 1 to 10. In certain embodiments, k is an integer from 1 to 9. In certain embodiments, k is an integer from 1 to 8. In certain embodiments, k is an integer from 1 to 7. In certain embodiments, k is an integer from 1 to 6. In certain embodiments, k is an integer from 1 to 5. In certain embodiments, k is an integer from 1 to 4. In certain embodiments, k is an integer from 1 to 3. In certain embodiments, k is an integer from 1 to 2.

In certain embodiments, p is an integer from 1 to 8000. In certain embodiments, p is an integer from 1 to 7000. In certain embodiments, p is an integer from 1 to 6000. In certain embodiments, p is an integer from 1 to 5000. In certain embodiments, p is an integer from 1 to 4000. In certain embodiments, p is an integer from 1 to 3000. In certain embodiments, p is an integer from 1 to 2000. In certain embodiments, p is an integer from 1 to 1000. In certain embodiments, p is an integer from 1 to 500. In certain embodiments, p is an integer from 1 to 400. In certain embodiments, p is an integer from 1 to 300. In certain embodiments, p is an integer from 1 to 200. In certain embodiments, p is an integer from 1 to 100. In certain embodiments, p is an integer from 1 to 90. In certain embodiments, p is an integer from 1 to 80. In certain embodiments, p is an integer from 1 to 70. In certain embodiments, p is an integer from 1 to 60. In certain embodiments, p is an integer from 1 to 50. In certain embodiments, p is an integer from 1 to 40. In certain embodiments, p is an integer from 1 to 30. In certain embodiments, p is an integer from 1 to 20. In certain embodiments, p is an integer from 1 to 10. In certain embodiments, p is an integer from 1 to 5.

In certain embodiments, n is an integer from 1 to 4000. In certain embodiments, n is an integer from 1 to 3000. In certain embodiments, n is an integer from 1 to 2000. In certain embodiments, n is an integer from 1 to 1000. In certain embodiments, n is an integer from 1 to 700. In certain embodiments, n is an integer from 1 to 800. In certain embodiments, n is an integer from 1 to 600. In certain embodiments, n is an integer from 1 to 500. In certain embodiments, n is an integer from 1 to 400. In certain embodiments, n is an integer from 1 to 300. In certain embodiments, n is an integer from 1 to 200. In certain embodiments, n is an integer from 1 to 100. In certain embodiments, n is an integer from 1 to 90. In certain embodiments, n is an integer from 1 to 80. In certain embodiments, n is an integer from 1 to 70. In certain embodiments, n is an integer from 1 to 60. In certain embodiments, n is an integer from 1 to 50. In certain embodiments, n is an integer from 1 to 40. In certain embodiments, n is an integer from 1 to 30. In certain embodiments, n is an integer from 1 to 20. In certain embodiments, n is an integer from 1 to 10. In certain embodiments, n is an integer from 1 to 5.

In certain embodiments, compounds of Formula III used in the methods of derivatization provided herein include, but are not limited to, amino sugars. In certain embodiments, such amino sugars include, but are not limited to, D-mannosamine and D-galactosamine.

In certain embodiments, compounds of Formula IV used in the derivatization methods provided herein include 2-amino-benzaldehyde (2-ABA), derivatives of 2-amino-benzaldehyde (2-ABA), 2- Derivatives of amino-benzaldehyde (2-ABA), 2-amino-acetophenone (2-AAP), derivatives of 2-amino-acetophenone (2-AAP), 2-amino-acetophenone provided herein (2- AAP), 2-amino-5-nitro-benzophenone (2-ANBP), 2-amino-5-nitro-benzophenone (2-ANBP) and 2-amino-5-nitro provided herein -Derivatives of benzophenone (2-ANBP) are included, but are not limited thereto.

Figure 22 shows the reaction scheme for the chemical derivatization of PCL-A with 2-amino-benzaldehyde (2-ABA), wherein at least one PCL-A has been site-specifically incorporated into the protein. The cyclization reaction of semialdehyde with 2-ABA forms a quinazoline-type moiety (22-A or 22-B) as a Friedlaender-type reaction, which is further reduced when using an appropriate reducing agent to form tetrahydrogen. Quinazoline type residues (22-D) or substituted anilines (22-E) can be formed. Alternatively, a fused ring moiety (22-C) is formed by further reaction of the quinazoline-type moiety 22-B. Suitable reducing agents for reducing the quinazoline-type moiety include, but are not limited to, sodium cyanoborohydride and sodium borohydride.

In addition, Figure 23 shows the protein conjugates of various structures formed after the reaction of PCL-A and 2-ABA, 2-AAP or 2-ANBP. The expected mass increase due to the attachment of these groups was also shown. The structure obtained using this group was characterized by NMR spectroscopy (see Example 45). In addition, NaCNBH ₃ reduction was found to stabilize PCL-based protein conjugates and prevent dissociation of PCL-ABA and PCL-AAP linkages even at high temperatures (see Example 44).

2-ABA and Δ ¹ -pyrroline-5-carboxylic acid (L-1-pyrroline-5-carboxylic acid) (Strecker, HJ, (1971), Methods in Enzymology 27B, 254-257), [Vogel, HJ, and Davis, BD (1953), "Glutamic gamma-semialdehyde and delta1-pyrroline-5-carboxylic acid, intermediates in the biosynthesis of proline," J. Am. Chem. Soc. 74, 109-112] And [Schoepf, C., and Oechler, F., (1936), Ann. Chem. 523, 1]) and other pyrrolines (Schoepf, C., and Oechler, F., (1936), Ann. ... Chem 523, 1] and [Schoepf, C., Steuer and, H., (1947), Ann Chem 558, 124] view) of the reaction is Δ ¹ in proline metabolism and biosynthesis-pyrroline-5-carboxylic It has been used as a colorimetric assay in studying the role of acids (Vogel, HJ, and Davis, BD (1953), "Glutamic gamma-semialdehyde and Δ ¹ -pyrroline-5-carboxylic acid, intermediates in the biosynthesis. of proline," J. Am. Chem. Soc. 74, 109-112], [Strecker, HJ, (1960), "The interconversion of glutamic acid and proline," J. Biol. Chem. 235, 2045-2050] , [Mezl, VA, and Knox, WE, (1976), "Properties and analysis of a stable derivative of pyrroline-5-carboxylic acid for use in metabolic studies," Analytical Bi ochemistry 74, 430-40], [Wu, GY, and Seifter, S., (1975), "A new method for the preparation of delta 1-pyrroline 5-carboxylic acid and proline," Analytical Biochemistry 67, 413-21 ], [Williams, I., and Frank, L., (1975), "Improved chemical synthesis and enzymatic assay of Δ ¹ -pyrroline-5-carboxylic acid," Anal. Biochem. 64, 85-97] and [Strecker, HJ, (1957), "The interconversion of glutamic acid and proline," J. Biol. Chem. 225, 825). The reaction scheme shows the formation of a glutamic acid-gamma-semialdehyde intermediate, but the reaction can proceed directly from pyrroline-carboxy-lysine without the formation of this intermediate.

A high-resolution mass spectrometric study of the model protein hRBP4 demonstrated chemical derivatization of PCL residues by 2-ABA, where PCL was site-specific incorporated into the protein hRBP4 (see FIGS. 25-26, Example 11). Figure 24 shows the mass spectrometric analysis of hRBP4 Phe122PCL derivatized with 2-ABA, wherein the protein derivatized with 2-ABA produced a major peak at 23269.2 Da, and the minimum amount of unmodified protein was at 23166.8 Da. Was detected. The increase in mass of 102.4 Da for the derivatized protein is consistent with the expected increase of 103 Da for the attachment of the 2-ABA residue, as shown in FIG. 23, so that the protein with site-specific incorporation of PCL is site-specific at the PCL site. It proves that it is transformed into an enemy. The MS/MS analysis result obtained after LC-MS analysis of the trypsin digest of 2-ABA-derivated hRBP4 Phe122PCL protein (FIG. 25) confirmed the expected YWGVASF*LQK peptide, where F* is 2-ABA- It has a mass consistent with the modified PCL. Figure 25A is the fragmentation pattern of the YWGVASF*LQK peptide, where F* has a mass consistent with the 2-ABA-modified PCL. Figure 25B is the TIC (total ion chromatogram) and EIC (extracted ion chromatogram) of the 2+ ion of YWGVASF*LQK (F* = PCL and PCL-2-ABA adduct), where derivatized species and derivatization Comparison of EIC to non-detectable (not detectable) species indicates completion of the reaction. Figure 25C is a mass spectrometric analysis of hRBP4 Phe122PCL derivatized with 2-ABA, with 3+ and 2+ precursors of YWGVASF*LQK (F* = PCL-2-ABA adduct) m/z 459.92 (3+) And 689.37 (2+), respectively, demonstrating that the reaction observed with 2-ABA occurs site-specific with the incorporated PCL residue at the desired TAG site at residue 122.

The evaluation of the pH dependence of derivatization is shown in FIG. The mass spectrum of hRBP4 (hRBP4 Phe122PCL) with PCL incorporated at position 122 is shown in Figure 26A, where hRBP4 Phe122PCL did not react with 2-ABA, and Figures 26B and 26C show hRBP4 Phe122PCL in acetate at pH 5.0. It is a mass spectrum after reaction with each of 10 mM 2-ABA in buffer and 10 mM 2-ABA in phosphate buffer at pH 7.4. The reaction of 2-ABA with the PCL residue of the hRBP4 protein proceeds rapidly at room temperature to a maximum of 95% completion in an aqueous buffer adjusted to pH 5 and to approximately 87% to a slightly lower degree at pH 7.4. As shown in Figures 27D to F, the reaction with 2-ABA is selective for the PCL residue, wherein hRBP4 labeled with an inactive non-natural amino acid O-methyl-phenylalanine (OMePhe) at position 62 and wild-type phenylalanine at position 122 (Phe ) In the case of hRBP4 having a residue, no reaction with 2-ABA was observed.

The evaluation of the reaction efficiency as a function of the reactant:protein concentration ratio and the reactivity with the 2-ABA-like reactant is shown in FIG. 27. The mass spectrum after reaction of hRBP4 (hRBP4 Phe122PCL) with PCL incorporated at position 122 with 0.1 mM 2-amino-benzaldehyde (2-ABA) is shown in Fig.27A, and 0.1 mM 2-amino-acetophenone of hRBP4 Phe122PCL ( 2-AAP) and 0.1 mM 2-amino-5-nitro-benzophenone (2-ANBP) are shown in Figs. 27B and 27C respectively. All reactions were carried out in 200 mM sodium acetate, pH 5.0. As in Figure 23, the protein conjugate was detected to have the expected mass increase at the correct mass. In addition, yields of greater than 88% conversion were achieved for 2-ABA and 2-AAP, but about 5% yield was achieved for 2-ANBP due to low solubility. However, this demonstrates that the 2-ABA analog reacts efficiently at the site of PCL incorporation when the reactant:protein concentration ratio is 6:1. The degree of reaction was only slightly lower than when the reactant:protein ratio was carried out at 600:1 (FIG. 26).

There was no significant improvement in the degree of derivatization when the derivatization agent was added in a final concentration range of 0.1 to 10 mM corresponding to a molar excess of 6 to 600 times compared to the protein. In addition, at molar ratios above 4700, additional protein residues expected to be lysine were derivatized by 2-ABA (FIG. 28). In Figure 28, a significant excess of 2-ABA compared to protein in 10×PBS is hRBP4 (A, about 17 μM, 4700-fold excess of 2-ABA compared to protein) and OMePhe incorporated hRBP4 (B, about 6.5 μM, compared to protein). 15400 fold excess) induced conjugation at additional sites, exemplifying that conjugation at these additional sites is mediated by residues other than PCL.

To further illustrate the utility of the methods provided herein, FIG. 29 shows before and after derivatization of PCL incorporated in FAS-TE with 2-amino-acetophenone (2-AAP) at pH 5.0 and pH 7.4. The mass spectrum is shown (see Example 12). Figure 29A shows the mass spectrum of the unreacted FAS-TE Tyr2454PCL, and Figure 29B shows the mass spectrum of the reaction mixture at pH 5.0. Here, 100% of the observable peak intensity occurs at 33318.8 Da, which is 116.8 Da greater than for the unreacted material, as expected a mass increase of 117 Da for the 2-AAP modified FAS-TE Tyr2454PCL. Similarly, at pH 7.4 the reaction proceeded to 95% completion (Fig. 29C (unreacted) and Fig. 29D (reacted)).

30 is a general reaction scheme for site-specific modification of a protein through chemical derivatization of pyrrolysine and/or PCL using 2-amino-benzaldehyde or 2-amino-benzaldehyde analogue. Certain embodiments of such analogs are provided herein. The reaction of 2-ABA with pyrrolysine and the pyrroline ring of PCL is ^{similar to that of Δ 1} -pyrroline-5-carboxylic acid and 2-ABA. Similarly, the reaction of pyrrolysine and PCL with 2-AAP and 2-ANBP results in a protein modified with each of the substituted residues in FIG. 23.

In certain embodiments, the 2-ABA functionality is used to site-specifically attach various groups to proteins, polypeptides and/or peptides with pyrrolysine or PCL incorporated therein. These groups include labels, dyes, polymers, water-soluble polymers, polyalkylene glycols, poly(ethylene glycol), derivatives of poly(ethylene glycol), sugars, lipids, photocrosslinkers, cytotoxic compounds, drugs, affinity labels, photoaffinities. Label, reactive compound; Resin, peptide, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelating agent, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, PCR probe, antisense polynucleotide, ribo-oligonucleotide, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labeling , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocausing moieties, actinic radiation excitable moieties, ligands, photoisomerizing moieties, biotin, biotin analogs , Heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-activators, amino thio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups , Chromophore group, chemiluminescent group, fluorescent moiety, electron high density group, magnetic group, intercalating group, chelating group, chromophore, energy transfer agent, biologically active agent, detectable label, small molecule, inhibitory ribonucleic acid, siRNA, radionucleotide , Neutron-capturing agent, biotin derivative, quantum dot(s), nanotransmitter, radiotransmitter, abzyme, enzyme, activated complex activator, virus, toxin, adjuvant, TLR2 agonist, TLR4 agonist, TLR7 Agonist, TLR9 agonist, TLR8 agonist, T-cell epitope, phospholipid, LPS-like molecule, keyhole limpet hemocyanin (KLH), immunogenic hapten, aglycan, allergen, angiostatin, antihormonal, antioxidant, Aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, mimotopes, receptors, reverse micelles, detergents, immune response enhancers, fluorescent dyes, FRET reagents, radiation-imaging probes, other spectroscopic probes, prodrugs, for immunotherapy Toxin, solid support, -CH ₂ CH ₂ -(OCH ₂ CH ₂ O) _p -OX ² ,- _{O- (CH 2 CH 2 O)} p CH 2 CH 2 -X 2, and any combination thereof (wherein, p is 1 to 10,000, X ² is H, C _{1 - 8} alkyl, protecting group or a terminal functional group Im) is included, but is not limited thereto.

In certain embodiments, the 2-AAP functionality is used to site-specifically attach various groups to proteins, polypeptides and/or peptides with pyrrolysine or PCL incorporated therein. These groups include labels, dyes, polymers, water-soluble polymers, polyalkylene glycols, poly(ethylene glycol), derivatives of poly(ethylene glycol), sugars, lipids, photocrosslinkers, cytotoxic compounds, drugs, affinity labels, photoaffinities. Label, reactive compound; Resin, peptide, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelating agent, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, PCR probe, antisense polynucleotide, ribo-oligonucleotide, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labeling , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocausing moieties, actinic radiation excitable moieties, ligands, photoisomerizing moieties, biotin, biotin analogs , Heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-activators, amino thio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups , Chromophore group, chemiluminescent group, fluorescent moiety, electron high density group, magnetic group, intercalating group, chelating group, chromophore, energy transfer agent, biologically active agent, detectable label, small molecule, inhibitory ribonucleic acid, siRNA, radionucleotide , Neutron-capturing agent, biotin derivative, quantum dot(s), nanotransmitter, radiotransmitter, abzyme, enzyme, activated complex activator, virus, toxin, adjuvant, TLR2 agonist, TLR4 agonist, TLR7 Agonist, TLR9 agonist, TLR8 agonist, T-cell epitope, phospholipid, LPS-like molecule, keyhole limpet hemocyanin (KLH), immunogenic hapten, aglycan, allergen, angiostatin, antihormonal, antioxidant, Aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, mimotopes, receptors, reverse micelles, detergents, immune response enhancers, fluorescent dyes, FRET reagents, radiation-imaging probes, other spectroscopic probes, prodrugs, for immunotherapy Toxin, solid support, -CH ₂ CH ₂ -(OCH ₂ CH ₂ O) _p -OX ² ,- _{O- (CH 2 CH 2 O)} p CH 2 CH 2 -X 2, and any combination thereof (wherein, p is 1 to 10,000, X ² is H, C _{1 - 8} alkyl, protecting group or a terminal functional group Im) is included, but is not limited thereto.

In certain embodiments, the 2-ANPA functionality is used to site-specifically attach various groups to proteins, polypeptides and/or peptides with pyrrolysine or PCL incorporated therein. These groups include labels, dyes, polymers, water-soluble polymers, polyalkylene glycols, poly(ethylene glycol), derivatives of poly(ethylene glycol), sugars, lipids, photocrosslinkers, cytotoxic compounds, drugs, affinity labels, photoaffinities. Label, reactive compound; Resin, peptide, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelating agent, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, PCR probe, antisense polynucleotide, ribo-oligonucleotide, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labeling , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocausing moieties, actinic radiation excitable moieties, ligands, photoisomerizing moieties, biotin, biotin analogs , Heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-activators, amino thio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups , Chromophore group, chemiluminescent group, fluorescent moiety, electron high density group, magnetic group, intercalating group, chelating group, chromophore, energy transfer agent, biologically active agent, detectable label, small molecule, inhibitory ribonucleic acid, siRNA, radionucleotide , Neutron-capturing agent, biotin derivative, quantum dot(s), nanotransmitter, radiotransmitter, abzyme, enzyme, activated complex activator, virus, toxin, adjuvant, TLR2 agonist, TLR4 agonist, TLR7 Agonist, TLR9 agonist, TLR8 agonist, T-cell epitope, phospholipid, LPS-like molecule, keyhole limpet hemocyanin (KLH), immunogenic hapten, aglycan, allergen, angiostatin, antihormonal, antioxidant, Aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, mimotopes, receptors, reverse micelles, detergents, immune response enhancers, fluorescent dyes, FRET reagents, radiation-imaging probes, other spectroscopic probes, prodrugs, for immunotherapy Toxin, solid support, -CH ₂ CH ₂ -(OCH ₂ CH ₂ O) _p -OX ² ,- _{O- (CH 2 CH 2 O)} p CH 2 CH 2 -X 2, and any combination thereof (wherein, p is 1 to 10,000, X ² is H, C _{1 - 8} alkyl, protecting group or a terminal functional group Im) is included, but is not limited thereto.

Attach any of these above mentioned groups to 2-ABA, 2-AAP and 2-ANPA using any of the attachment points R ₇ to R _{11 shown in FIG. 30.} In other embodiments, the benzene ring is replaced with any other ring structure provided herein, including but not limited to naphthalene. In other embodiments, the benzene ring is replaced with a sugar.

The concentration of the derivatizing agent used in the methods provided herein for the derivatization of pyrrolysine and PCL incorporated into proteins, polypeptides and/or peptides ranges from about 0.005 mM to about 50 mM. In certain embodiments, the concentration of the derivatizing agent used in the methods provided herein for derivatization of pyrrolysine or PCL ranges from about 0.005 mM to about 25 mM. In certain embodiments, the concentration of the derivatizing agent used in the methods provided herein for derivatization of pyrrolysine or PCL ranges from about 0.005 mM to about 10 mM. In certain embodiments, the concentration of the derivatizing agent used in the methods provided herein for derivatization of pyrrolysine or PCL ranges from about 0.005 mM to about 5 mM. In certain embodiments, the concentration of the derivatizing agent used in the methods provided herein for the derivatization of pyrrolysine or PCL ranges from about 0.005 mM to about 2 mM. In certain embodiments, the concentration of the derivatizing agent used in the methods provided herein for derivatization of pyrrolysine or PCL ranges from about 0.005 mM to about 1 mM.

In the derivatization methods provided herein, the molar ratio of the derivatizing agent to the protein, polypeptide or peptide having at least one pyrrolysine or PCL incorporated therein ranges from about 0.05 to about 10000. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 8000. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 6000. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 4000. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 2000. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 1000. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 800. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 600. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 400. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 200. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 100. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 50. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 25. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 10. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 0.05 to about 1.

In other embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 10000. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 8000. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 6000. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 4000. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 2000. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 1000. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 800. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 600. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 400. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 200. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 100. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 50. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 25. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 1.5 to about 10. In certain embodiments, the molar ratio of derivatizing agent to such protein, polypeptide or peptide ranges from about 6 to about 600.

The pH of the buffer solution used for the derivatization of proteins, polypeptides and/or peptides using the methods and compositions provided herein is from about pH 2 to about pH 10. In certain embodiments, the pH is about pH 4 to about pH 8. In certain embodiments, the pH is about pH 4 to about pH 7.5. In certain embodiments, the pH is about pH 4 to about pH 7. In certain embodiments, the pH is about pH 4 to about pH 6.5. In certain embodiments, the pH is about pH 4 to about pH 6. In certain embodiments, the pH is about pH 4 to about pH 5.5. In certain embodiments, the pH is about pH 4 to about pH 5. In certain embodiments, the pH is about pH 4 to about pH 4.5. In certain embodiments, the pH is about pH 6 to about pH 8. In certain embodiments, the pH is about pH 6 to about pH 7.5. In certain embodiments, the pH is about pH 6 to about pH 7. In certain embodiments, the pH is about pH 6 to about pH 6.5. In certain embodiments, the pH is about pH 7 to about pH 8. In certain embodiments, the pH is about pH 7 to about pH 7.5.

The derivatization method provided herein is useful for the derivatization of pyrrolysine or PCL site-specifically incorporated into a protein. In certain embodiments, such methods of derivatization are used to derivatize a PCL residue incorporated at a single site into a protein, polypeptide and/or peptide. In other embodiments, such methods of derivatization are used to derivatize PCL residues incorporated at multiple sites into proteins, polypeptides and/or peptides. In certain embodiments, the protein, polypeptide or peptide derivatized using the methods and compositions provided herein is 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15 or more pyrrolysines or pyrrolysine analogs.

In another aspect provided herein, proteins, polypeptides and peptides having at least one pyrrolysine or PCL incorporated therein are derivatized by at least one simultaneous or post-translational modification. Non-limiting examples of such simultaneous or post-translational modifications include glycosylation, acetylation, acylation, methylation, nitration, sulfation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like. Included, but not limited to. Also included in this aspect are methods of producing, purifying, characterizing and using such proteins, polypeptides and peptides containing at least one such simultaneous or post-translational modification.

Biotherapeutics with site-specific modification

PCL incorporated into protein, polypeptide and/or peptide and coupled to pyrrolysine Macromolecular polymer

In certain embodiments, macromolecular polymers are added to pyrrolysine or PCL residues incorporated into proteins, polypeptides and/or peptides using the compositions, methods, techniques and strategies described herein. A wide variety of macromolecular polymers can be coupled to pyrrolysine and PCL residues incorporated into the proteins, polypeptides and/or peptides described herein. Such modifications are used to modulate the biological properties of such proteins, polypeptides and/or peptides and/or provide new biological properties to such proteins, polypeptides and/or peptides. In certain embodiments, the macromolecular polymer is coupled to such proteins, polypeptides and/or peptides via direct coupling to the pyrrolysine or PCL residue(s) incorporated in the proteins, polypeptides and/or peptides provided herein. . In other embodiments, the macromolecular polymer is coupled to proteins, polypeptides and/or peptides provided herein via bifunctional, trifunctional, tetrafunctional and multifunctional linkers coupled to pyrrolysine or PCL residue(s). do. In other embodiments, the macromolecular polymer is coupled to the proteins, polypeptides and/or peptides provided herein via a bifunctional linker coupled to a pyrrolysine or PCL residue(s). In certain embodiments, such bifunctional, trifunctional, tetrafunctional and polyfunctional linkers are monofunctional linkers whose all ends are specific substituents for reaction with pyrrolysine or PCL moieties. In certain embodiments, such bifunctional, trifunctional, tetrafunctional and multifunctional linkers have at least one end being a substituent specific for reaction with pyrrolysine or PCL residue(s), and the other end is pyrrolysine or PCL. Another functional substituent that does not react with the moiety(s) is a heterobifunctional linker. Certain substituents of pyrrolysine or PCL-specific reactive groups are provided herein, and other functional substituents and resulting linkages used in such bifunctional, trifunctional, tetrafunctional and multifunctional linkers include those described in Table 1, It is not limited.

Covalent attachment of macromolecular polymers to biologically active molecules, such as proteins, polypeptides and/or peptides provided herein, increases water solubility (e.g., water solubility in a physiological environment), increases bioavailability, increases serum half-life. An approach for increasing, increasing the therapeutic half-life, modulating immunogenicity, modulating biological activity, or prolonging the circulation time of such biologically active molecules. Important features of such macromolecular polymers include biocompatibility, non-toxicity, and absence of immunogenicity, and treatment of proteins, polypeptides and/or peptides provided herein coupled to the macromolecular polymer via a pyrrolysine or PCL moiety. For use these macromolecular polymers are pharmaceutically acceptable.

Certain macromolecular polymers coupled to pyrrolysine or PCL residue(s) incorporated into the proteins, polypeptides and/or peptides described herein are water soluble polymers. In certain embodiments, such water soluble polymers are coupled via pyrrolysine or PCL residue(s) incorporated into proteins, polypeptides and/or peptides provided herein, and in other embodiments such water soluble polymers are provided herein. , Pyrrolysine incorporated into the polypeptide and/or peptide, or any functional group or substituent coupled to the PCL residue(s). In some embodiments, proteins, polypeptides and/or peptides provided herein comprise one or more PCL residues coupled to a water soluble polymer and one or more naturally occurring amino acids linked to a water soluble polymer.

Structural forms of macromolecular polymers coupled to pyrrolysine or PCL residues incorporated into proteins, polypeptides and/or peptides include, but are not limited to, linear, forked or branched polymers. In certain embodiments, the backbone of such a branched or forked water soluble polymer has from 2 to about 300 ends. In certain embodiments, each end of such a polyfunctional polymer derivative, such as, but not limited to, a linear polymer having two ends comprises a functional group. In certain embodiments, these functional groups are the same, and in other embodiments, these functional groups are different. Non-limiting examples of such terminal functional groups include N-succinimidyl carbonate, amine, hydrazide, succinimidyl propionate and succinimidyl butanoate, succinimidyl succinate, succinimidyl ester, benzotria Sol carbonate, glycidyl ether, oxycarbonylimidazole, p-nitrophenyl carbonate, aldehyde, maleimide, orthopyridyl-disulfide, acrylol, and vinylsulfone. Does not. In other embodiments, functional groups include those listed in Table 1.

The covalent attachment of a water-soluble polymer (also referred to herein as a hydrophilic polymer) to a biologically active molecule, such as a protein, polypeptide and/or peptide provided herein, is a water solubility (e.g., water solubility in a physiological environment). Of, increase in bioavailability, increase in serum half-life, increase in therapeutic half-life, modulate immunogenicity, modulate biological activity, or prolong the circulation time of such biologically active molecules. Important features of such water-soluble polymers include biocompatibility, non-toxicity, and absence of immunogenicity, and for therapeutic use of proteins, polypeptides and/or peptides provided herein coupled to water-soluble polymers via pyrrolysine or PCL moieties. For this, these macromolecular polymers are pharmaceutically acceptable.

Hydrophilic polymers coupled to proteins, polypeptides and/or peptides via pyrrolysine or PCL moieties include polyalkyl ethers and alkoxy-capped analogs thereof, polyvinylpyrrolidone, polyvinylalkyl ether, polyoxazoline, polyalkyl. Oxazoline, polyhydroxyalkyl oxazoline, polyacrylamide, polyalkyl acrylamide, polyhydroxyalkyl acrylamide, polyhydroxyalkyl acrylate, polysialic acid and its analogs, hydrophilic peptide sequences; Polysaccharides and derivatives thereof, cellulose and derivatives thereof, chitin and derivatives thereof, hyaluronic acid and derivatives thereof, starch, alginate, chondroitin sulfate, albumin, pullulan, carboxymethyl pullulan, polyamino acids and derivatives thereof, maleic acid Anhydride copolymers, polyvinyl alcohol and copolymers thereof, polyvinyl alcohol and terpolymers thereof, and combinations thereof.

Such polyalkyl ethers and alkoxy-capped analogs thereof include polyoxyethylene glycol (also known as poly(ethylene glycol) or PEG), polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogs thereof. Includes, but is not limited to. Such polyhydroxyalkyl acrylamides include, but are not limited to, polyhydroxypropylmethacrylamide and derivatives thereof. Such polysaccharides and derivatives thereof include, but are not limited to, dextran and dextran derivatives (eg (by way of example only), carboxymethyldextran, dextran sulfate and aminodextran). Such cellulose and its derivatives include, but are not limited to, carboxymethyl cellulose and hydroxyalkyl cellulose. Such chitin and its derivatives include, but are not limited to, chitosan, succinyl chitosan, carboxymethylchitin, and carboxymethylchitosan. Such polyamino acids and derivatives thereof include, but are not limited to, polyglutamic acid, polylysine, polyaspartic acid and polyaspartamide. Such maleic anhydride copolymers include, but are not limited to, styrene maleic anhydride copolymers and divinylethyl ether maleic anhydride copolymers.

In some embodiments, a water-soluble polymer coupled directly or indirectly to such protein, polypeptide and/or peptide via pyrrolysine or PCL residue(s) incorporated into the proteins, polypeptides and/or peptides provided herein is a poly (Ethylene glycol) (PEG). Poly(ethylene glycol) is considered biocompatible, and PEG can coexist with it without causing harm to living tissues or organisms. PEG is also substantially non-immunogenic and therefore does not tend to elicit an immune response in the body. PEG is a hydrophilic polymer that has been widely used in pharmaceuticals, artificial implants, and other applications where biocompatibility, non-toxicity, and absence of immunogenicity are important. PEG conjugates tend not to elicit a substantial immune response or cause blood clotting or other undesirable effects. Thus, when attached to a molecule that has some desirable function in the body, such as a biologically active agent, PEG tends to mask the agent and can reduce or eliminate any immune response so that the organism will allow the presence of the agent. Make it possible.

In some embodiments, poly(ethylene glycol) coupled directly or indirectly to such protein, polypeptide and/or peptide via pyrrolysine or PCL residue(s) incorporated into the proteins, polypeptides and/or peptides provided herein. This is a straight chain polymer, and in other embodiments this PEG polymer is a branched polymer. The molecular weight or molecular weight distribution of these straight chain and branched PEG polymers ranges from about 100 Da to about 100,000 Da or higher. In some embodiments, the molecular weight or molecular weight distribution of such PEG polymers is between about 100 Da and 50,000 Da. In some embodiments, the molecular weight or molecular weight distribution of such PEG polymers is between about 100 Da and 40,000 Da. In some embodiments, the molecular weight or molecular weight distribution of such PEG polymers is between about 1,000 Da and 40,000 Da. In some embodiments, the molecular weight or molecular weight distribution of such PEG polymers is between about 5,000 Da and 40,000 Da. In some embodiments, the molecular weight or molecular weight distribution of such PEG polymers is between about 10,000 Da and 40,000 Da. In certain embodiments, the molecular weight of such PEG polymers is 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da , 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da or 100 Da.

Structural forms of PEG polymers coupled to pyrrolysine or PCL residue(s) incorporated into proteins, polypeptides and/or peptides include, but are not limited to, linear, forked or branched. In certain embodiments, the backbone of such branched or forked PEG polymers has from 2 to about 300 ends. In certain embodiments, each end of such a polyfunctional polymer derivative, such as, but not limited to, a linear polymer having two ends comprises a functional group. In certain embodiments, these functional groups are the same, and in other embodiments, at least one of these functional groups is different. In other embodiments, these functional groups are different.

In certain embodiments, at least one end of the PEG polymer comprises a functional group prior to coupling with the protein, polypeptide and/or peptide. In certain embodiments, PEG is a linear polymer having a functional group at one end, and in other embodiments, PEG is a linear polymer having a functional group at each end to form a bifunctional PEG polymer. In other embodiments, the PEG is a forked polymer having a functional group at one end, and in other embodiments, the PEG is a forked polymer having a functional group at two or more ends. In other embodiments, the PEG is a forked polymer having a functional group at each end to form a multifunctional PEG polymer. In other embodiments, the PEG is a branched polymer having a functional group at one end, and in other embodiments, the PEG is a branched polymer having a functional group at two or more ends. In other embodiments, the PEG is a branched polymer having a functional group at each end to form a multifunctional PEG polymer. In certain embodiments of these aforementioned PEG polymers the functional groups are the same, and in other embodiments of these functional groups at least one functional group is different. In other embodiments of these aforementioned PEG polymers, the functional groups are different. However, at least one end of the PEG polymer is available for reaction with at least one pyrrolysine or PCL moiety incorporated in the protein, polypeptide and/or peptide.

Non-limiting examples of terminal functional groups include N-succinimidyl carbonate, amine, hydrazide, succinimidyl propionate, succinimidyl butanoate, succinimidyl succinate, succinimidyl ester, benzotriazole Carbonate, glycidyl ether, oxycarbonylimidazole, p-nitrophenyl carbonate, aldehyde, maleimide, orthopyridyl-disulfide, acrylol, vinylsulfone, activated carbonate (e.g. For example (but not limited to), p-nitrophenyl ester), activated ester (e.g., but not limited to), N-hydroxysuccinimide, p-nitrophenyl ester), oxime, carbonyl , Dicarbonyl, hydroxylamine, hydroxyl, methoxy, benzaldehyde, acetophenone, 2-amino-benzaldehyde, 2-amino-acetophenone and 2-amino-5-nitro-benzophenone. Does not.

FIG. 31 is an embodiment of a functionalized PEG polymer coupled to the protein via at least one PCL residue incorporated into the protein, 2-amino-acetophenone-PEG ₈ (2-AAP-PEG8; TU3205-044). Illustratively. As shown, the 2-amino-acetophenone residue of 2-AAP-PEG8 forms a quinazoline-type residue as a spacer between _{PEG 8 and the protein.} These quinazoline-type moieties can further react to form a fused ring structure. In the case of the coupling of 2-AAP-PEG8, this derivatization adds 556 Da to the mass of the protein. Other functionalized PEGs used in the methods provided herein include, but are not limited to, those described in Example 20.

Figure 32 shows the mass spectrum of hRBP4 (hRBP4 Phe122PCL) with PCL incorporated at position 122 before and after derivatization with 2-AAP-PEG8 at pH 7.5 (Figure 32A) and pH 5.0 (Figure 32B). . This coupling reaction was carried out at pH 7.5 and pH 5, where 89% to 100% conversion occurred, resulting in an expected mass increase of 556 Da versus unreacted protein (FIG. 32C ). Wild-type hRBP4 did not react with a 450-fold or 2300-fold excess of 2-AAP-PEG8 (FIGS. 32D-F). Since no coupling occurs in the absence of PCL, this further illustrates the specificity of the coupling reaction between PCL and 2AAP-PEG8.

To further illustrate the utility of this labeling method, the thioesterase domain of human fatty acid synthetase (FAS-TE) (FAS-TE Tyr2454PCL) with PCL incorporated at position 2454, produced in Escherichia coli It was derivatized with 2-AAP-PEG8 (see Figure 33, Example 14). Figure 33A shows the mass spectrum (mass 33202 Da) of the unreacted protein, and Figure 33B shows the mass spectrum of FAS-TE Tyr2454PCL having an expected mass of 33756 Da by reacting with 2-AAP-PEG8 (TU3205-044) until completion. Show. Additional examples are provided in Figures 33C and 33D, which are FAS-TE with PCL incorporated at position 2454 (FAS-TE Tyr2454PCL) produced in Escherichia coli at room temperature (Figure 33C) and at 4°C. (Fig. 33D) It shows the mass spectrum after derivatization with 2.4 kDa 2-AAP-PEG (TU3205-048). Under the conditions used, only approximately 25% conversion was achieved, yielding a protein with an expected mass of 35585 Da.

In addition, Figure 34 shows the PEGylation of FAS-TE Tyr2454PCL using 2.4 kDa 2-AAP-PEG and 23 kDa 2-AAP-PEG in the molar ratio shown in the figure. The 0.5 kDa 2-AAP-PEG (2-AAP-PEG8) PEGylated product was not separated on SDS-PAGE, but was verified by mass spectrometry.

After incorporation of PCL at 20 positions into fibroblast growth factor 21 (FGF-21) using the method provided herein, the corresponding PCL residues were PEGylated (see Example 15). Figure 35 shows mass spectra obtained before and after derivatization of FGF21 Lys84PCL with 2-AAP-PEG8. Fig. 35A is a mass spectrum of unreacted FGF21 Lys84PCL (21235.6 Da), and Fig. 35B is a mass spectrum of FGF21 Lys84PCL reacted with 2-AAP-PEG8. The PEGylation reaction proceeded to completion, resulting in a protein of 21792.4 Da. The increase of 556.8 Da is consistent with the expected increase of 556 Da for this derivatization. Figure 36 shows the SDS-PAGE results obtained after derivatization of 7 kinds of FGF21 PCL mutants with 23 kDa 2-AAP-PEG. Figure 36A is an SDS gel of a reaction mixture of FGF21 PCL mutant (0.1 to 0.4 mM) reacted with 23 kDa 2-AAP-PEG (pH 7.4, 4° C., 60 hours), PEG-FGF21, full length (FL) FGF21- PCL and truncated (TR) FGF21-PCL are shown. Figure 36B shows the SDS-PAGE results of eight FGF21 PCL mutants after partial purification of PEG-FGF21, full length (FL) and truncated (TR) FGF21.

After incorporation of PCL at 11 positions into mouse erythropoietin (EPO) using the method provided herein, the corresponding PCL residues were PEGylated for the three EPO PCL mutants (see Example 6). 37 is an SDS gel obtained after PEGylation of mouse EPO PCL mutants. Mouse EPO with PCL incorporated at three different positions in HEK293F cells was reacted with 2-AAP-mPEG23k (TU3205-052). PEGylated EPO and non-PEGylated EPO are indicated by arrows.

In certain embodiments, the bifunctional or polyfunctional PEG polymer linked to the protein, polypeptide and/or peptide via PCL residue(s) is further derivatized or substituted with the remaining functional groups on the unreacted end. In certain embodiments, a bifunctional or polyfunctional PEG polymer linked to a protein, polypeptide and/or peptide via reaction of a benzaldehyde, benzophenone or acetophenone moiety with a PCL moiety(s) is added using the remaining functional groups on the unreacted end. Derivatized or substituted with.

The methods and compositions described herein provide a highly efficient method for the selective modification of proteins, polypeptides and peptides using PEG derivatives, which are suitably incorporated after selectively incorporating the amino acid PCL or pyrrolysine into the protein in response to a selector codon. It involves modifying the corresponding amino acid residues using functionalized PEG derivatives. Accordingly, provided herein are proteins, polypeptides and/or peptides having PCL or pyrrolysine residue(s) incorporated therein, and also herein is a water-soluble polymer such as poly(s) via such PCL or pyrrolysine residue(s). Such proteins, polypeptides and/or peptides linked to (ethylene glycol) (PEG) are provided.

The degree of PEGylation of the proteins, polypeptides and/or peptides described herein (i.e., the number of PEG polymers linked to the protein, polypeptide and/or peptide) is adjustable to provide altered pharmacological, pharmacokinetic or pharmacodynamic characteristics. In certain embodiments this change is an increase in this characteristic, and in other embodiments this change is a decrease in this characteristic. In certain embodiments, this characteristic is an in vivo half-life. In some embodiments, the half-life of a protein, polypeptide and/or peptide PEGylated using the methods and compositions provided herein is at least about 10%, 20%, 30%, 40 compared to the unmodified protein, polypeptide and/or peptide. %, 50%, 60%, 70%, 80%, 90%, 2 times, 5 times, 10 times, 50 times, 100 times or at least about 200 times.

In certain embodiments, proteins, polypeptides and/or peptides PEGylated using the methods and compositions provided herein may be selected from hydrophobic chromatography, affinity chromatography; Anion exchange chromatography, cation exchange chromatography, Q quaternary ammonium resin, DEAE SEPHAROSE, chromatography on silica; Reverse phase HPLC, gel filtration (e.g., but not limited to), SEPHADEX G-75, Sephacryl S-100, Superdex 75, 100 and 200 used), hydrophobic interaction Action chromatography, size-exclusion chromatography, metal-chelate chromatography; Ultrafiltration/diafiltration, ethanol precipitation, chromatographic focusing, alternative chromatography, electrophoretic procedures (e.g., but not limited to), preparative equipotential focusing), differential solubility (e.g., but not limited to ), ammonium sulfate precipitation) and extraction.

In another aspect, the PEG polymers used in the methods provided herein have weak or degradable linkages within the backbone. By way of example only, such a PEG polymer has an ester linkage to be hydrolyzed in the polymer backbone, and this hydrolysis cleaves the linkage to release the protein, polypeptide or peptide to which the PEG is attached.

In another aspect, the methods and compositions described herein are used to produce a substantially homogeneous formulation of polymer-protein conjugates. As used herein, “substantially homogeneous” means that polymer:protein conjugate molecules are observed to be more than half of the total protein. Polymer:protein conjugates have biological activity, and the "substantially homogeneous" PEGylated polypeptide formulations of the invention provided herein have the advantage of a homogeneous formulation, for example, lot by lot to lot) It is a sufficiently homogeneous formulation to show ease of clinical use in predicting pharmacokinetics.

In another aspect, the methods and compositions described herein are used to produce a substantially homogeneous formulation of polymer:protein conjugates. As used herein, “substantially homogeneous” means that polymer:protein conjugate molecules are observed to be more than half of the total protein. Polymer:protein conjugates have biological activity, and the "substantially homogeneous" PEGylated polypeptide formulations of the invention provided herein have the advantages of a homogeneous formulation, such as, by way of example only, prediction of pharmacokinetics per product number. It is a sufficiently homogeneous formulation to show ease in clinical use.

In another aspect, the methods and compositions described herein are used to prepare a mixture of polymer-protein conjugate molecules, and an advantage provided herein is that the ratio of mono-polymer:protein conjugates to be included in the mixture is selectable. Thus, in certain embodiments, mixtures of various proteins to which various numbers of polymeric moieties (i.e., non-, tri-, tetra-, etc.) are attached are prepared, and these conjugates are mono-polymers prepared by the methods described herein. It is optionally combined with a protein conjugate to produce a mixture having a proportion of the mono-polymer:protein conjugate.

The ratio of poly(ethylene glycol) molecules to protein molecules will depend on their concentration in the reaction mixture. In certain embodiments, the ratio of optimal (optimal in terms of reaction efficiency with minimal excess unreacted protein or polymer) is determined by the molecular weight of the selected poly(ethylene glycol) and the number of reactive groups available. The higher the molecular weight of the polymer, the fewer polymer molecules attached to the protein. Similarly, in other embodiments, the branching state of the PEG polymer is taken into account when optimizing these parameters. In this example, the higher the molecular weight (or the higher the number of branches), the higher the polymer:protein ratio.

Direct coupling of amino sugars with pyrrolysine and/or PCL incorporated into proteins, polypeptides and/or peptides

In another aspect provided herein, proteins, polypeptides and/or peptides having one or more pyrrolysine and/or PCL residue(s) incorporated therein are derivatized with amino sugars. 38 shows a general reaction scheme in which D-mannosamine is coupled to a protein with PCL incorporated therein ( _{illustrated as R 1 ).} In this embodiment, the aminoaldehyde residue of D-mannosamine reacts with the PCL residue to increase the mass of the protein by 161 Da. Other amino sugars used in this embodiment include, but are not limited to, mannosamine, galactosamine and glucosamine. Figure 39 shows the mass spectrum after the reaction of hRBP4 (hRBP4 Phe122PCL) with PCL incorporated at position 122 with mannosamine, and Figure 40 is a human fatty acid synthetase (FAS-TE) with PCL incorporated at position 2222 The mass spectra of (FAS-TE Leu2222PCL/Leu2223Ile) before (Fig. 40A) and after (Fig. 40B) reaction with mannosamine are shown.

Glycosylation of such proteins, polypeptides and/or peptides via coupling with pyrrolysine and PCL incorporated into proteins, polypeptides and/or peptides

The methods and compositions described herein are used to obtain proteins, polypeptides and peptides having one or more pyrrolysine and/or PCL residues bearing saccharide residues. The saccharide moiety is natural (e.g., but not limited to), N-acetylglucosamine and D-mannosamine) or non-natural (e.g., but not limited to), 3-fluorogalactose) Can be In certain embodiments, the saccharide is linked to pyrrolysine and/or PCL residues by non-natural linkages, including but not limited to the formation of quinazoline-type residues. In certain embodiments, the saccharide is linked to the pyrrolysine and/or PCL moieties by non-natural linkages, including but not limited to the formation of quinazoline-type moieties that have been further reduced with a reducing agent. In certain embodiments, the saccharide is pyrrolysine by non-natural linkage, including, but not limited to, the formation of quinazoline-type moieties that further react as provided herein to form a fused ring system. And/or to a PCL residue (see FIGS. 22, 23 and 30). In certain embodiments, the addition of saccharide(s), including but not limited to, the addition of glycosyl residues to proteins, polypeptides and peptides having one or more pyrrolysines and/or PCL incorporated therein It occurs in vivo. In other embodiments, the addition of saccharide(s), including but not limited to, the addition of glycosyl residues to proteins, polypeptides and peptides having one or more pyrrolysines and/or PCL incorporated therein Happens in vitro. In other embodiments, the saccharide once attached to the pyrrolysine and/or PCL moiety is further modified by glycosyltransferase and/or other enzymatic treatment to incorporate at least one pyrrolysine and/or PCL It produces an oligosaccharide bound to a protein, polypeptide or peptide having

FIG. 41 illustrates an embodiment in which simultaneous or post-translational modifications include attachment of an oligosaccharide to a protein, polypeptide or peptide through the formation of a quinazoline-type moiety. In certain embodiments, the saccharide is linked to the pyrrolysine and/or PCL moieties by non-natural linkages, including but not limited to the formation of quinazoline-type moieties that have been further reduced with a reducing agent. In certain embodiments, the saccharide is pyrrolysine by non-natural linkage, including, but not limited to, the formation of quinazoline-type moieties that further react as provided herein to form a fused ring system. And/or to a PCL residue (FIGS. 22, 23 and 30 ). In this embodiment, by way of example only, the oligosaccharide is linked to a 2-amino-acetophenone (2-AAP) conjugated to a pyrrolysine and/or PCL residue incorporated into a protein, polypeptide or peptide (GlcNAc- Man) ₂ -Man-GlcNAc-GlcNAc core. In another embodiment, the oligosaccharide comprises a (GlcNAc-Man) ₂ -Man-GlcNAc-GlcNAc core linked to 2-amino-benzaldehyde (2-ABA), and incorporated into a protein, polypeptide or peptide with 2-ABA. Protein conjugates are formed by reaction of pyrrolysine and/or PCL residues. In other embodiments, the oligosaccharide comprises a (GlcNAc-Man) ₂ -Man-GlcNAc-GlcNAc core linked to 2-amino-benzophenone (2-ABP), and is incorporated into a protein, polypeptide or peptide with 2-ABP. Protein conjugates are formed by reaction of pyrrolysine and/or PCL residues. Other oligosaccharides used in this embodiment have 2-ABA, 2-AAP or 2-ANBP residues.

By the covalent attachment of the protein alignment, heterodimer dimer, trimer yijongsam, two kinds of oligomers, such paper-mer, homologous trimer, oligomer homogeneous, asymmetric homodimer, forming a homogeneous asymmetric trimer and asymmetric homogeneous oligomer

In another aspect of using the methods provided herein, heterodimers, heterotrimers, heteromultimers, using site-specific incorporation of one or more pyrrolysines and/or PCLs into proteins, polypeptides and/or peptides, It forms protein-protein conjugates, including, but not limited to, homodimers, homotrimers, homomultimers, asymmetric homodimers, asymmetric homotrimers and asymmetric homomultimers. This protein-protein conjugate formation is carried out using site-specific crosslinking between proteins with pyrrolysine and PCL residue(s) incorporated therein. Figure 42 illustrates a specific embodiment (heterodimer, heterotrimer, homotrimer) of such protein-protein conjugate formation, in which proteins with PCL residue(s) incorporated therein are crosslinked. In Figure 42, the protein conjugate linkage formed by such crosslinking is a quinazoline-type residue that connects proteins together, but in other embodiments, the linkage is a reduced form of a quinazoline-type residue (Figs. 22, 23 and 30). In other embodiments, the linkage is a fused ring moiety (FIGS. 22, 23 and 30 ).

Non-limiting examples of such protein-protein conjugates include, but are not limited to: protein-protein conjugate cytokines, growth factors, growth factor receptors, interferons, interleukins, inflammatory molecules, oncogene products, peptide hormones, signals. Introducing molecule, steroid hormone receptor, transcription activator, transcription inhibitor, erythropoietin (EPO), fibroblast growth factor, fibroblast growth factor 21 (FGF21), leptin, insulin, human growth hormone, epithelial neutrophil activating peptide-78 , GROα/MGSA, GROβ, GROγ, MIP-1α, MIP-16, MCP-1, hepatocyte growth factor, insulin-like growth factor, leukemia inhibitory factor, oncostatin M, PD-ECSF, PDGF, pleiotropin, SCF, c-kit ligand, VEGF, G-CSF, IL-1, IL-2, IL-8, IGF-I, IGF-II, FGF (fibroblast growth factor), PDGF, TNF, TGF-α, TGF -ß, EGF (epidermal growth factor), KGF (keratinocyte growth factor), CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, hyalurin/CD44, Mos, Ras, Raf, Met ; p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and/or corticosterone. In another set of embodiments, the protein is homologous to the following therapeutic protein or other protein: alpha-1 anti-trypsin, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, Atrial natriuretic polypeptide, atrial peptide, CXC chemokine, T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4 , MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein- 1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, C-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine , Epithelial neutrophil activating peptide-78, GROα/MGSA, GROβ, GROγ, MIP-1α, MIP-16, MCP-1, epidermal growth factor (EGF), epithelial neutrophil activating peptide, exfoliation toxin, factor IX, factor VII, factor VIII, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, G-CSF, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, hedgehog protein, hemoglobin, hepatocyte Growth factor (HGF), hirudin, human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, Interferon, IFN-α, IFN-ß, IFN-γ, interleukin, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neuroturin, Neutrophil inhibitor (NIF), oncostatin M, bone morphogenetic protein, oncogene product, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, human growth hormone, pleiotropin, protein A, protein G, pyrogenic exotoxin A , B or C, relaxin, renin, SCF, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigen, staphylo Staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, Tumor growth factor (TGF), TGF-α, TGF-ß, tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular endothelial growth Factor (VEGF), urokinase, Mos, Ras, Raf, Met; p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and/or corticosterone.

Protein-protein conjugates, including but not limited to heterodimers, heterotrimers, heteromultimers, homodimers, homotrimers, homomultimers, asymmetric homodimers, asymmetric homotrimers and asymmetric homomultimers The crosslinking agent used for the formation of has a benzaldehyde, acetophenone and/or benzophenone residue at each end. These residues react with the pyrrolysine and/or PCL residue(s) incorporated into the protein using the methods provided herein, and such proteins include those provided herein. A non-limiting example of a bifunctional crosslinking agent used in the formation of a homodimer is shown in Fig. 43A. Fibroblast growth factor 21 (FGF-21) (FGF21 Lys84PCL) with a PCL residue incorporated at position 84 was crosslinked using this bifunctional linker. Figure 43B is the mass spectrum of the reaction mixture, where the peak for the covalent FGF21 Lys48PCL dimer with an expected mass of 43037.2 Da is the dominant species. Unreacted FGF21 Lys84PCL is detected at 21235.2 Da, but some monomeric FGF21 modified to one end of the attached crosslinker is detected at 21820.0 Da. By modifying the reaction conditions, FGF21 Lys84PCL was completely reacted. In FIG. 43 the protein conjugate linkage formed by such crosslinking is a quinazoline-type residue, but in other embodiments the linkage is a reduced form of a quinazoline-type residue (FIGS. 22, 23 and 30 ). In other embodiments, the linkage is a fused ring moiety (FIGS. 22, 23 and 30 ).

44A shows the mass spectrum of the reaction mixture, where the peak of the covalent FGF21 dimer with expected mass of 43034 Da is the dominant species. Unreacted FGF21 Lys84PCL is not detected at 21234 Da, but some monomeric FGF21 modified to one end of the attached crosslinker is detected at 21818 Da. 43B is SDS-PAGE for the reaction at pH 5.0 and pH 7.5, showing better conversion at pH 5.0. A dimer of FGF21 was generated using PCL-specific crosslinking. However, the method is applicable to any of the proteins described above and can be used to form dimers, trimers and multimers. This cross-linking is used to enhance the activity of biologically active proteins. In addition, this approach is used to stabilize complexes and/or to stabilize receptor decoys for structural studies. 45 shows an embodiment in which a crosslinking agent is used to form a trimer.

Site specific labeling

In another aspect of using the methods provided herein, site-specific incorporation of one or more pyrrolysines and/or PCLs into proteins, polypeptides and/or peptides is used to site-specificize such proteins, polypeptides and/or peptides. Labeled as a target. This labeling is due to the reaction of a label derivatized with a functional group that selectively reacts with pyrrolysine and/or PCL residue(s) with pyrrolysine and/or PCL residue(s). Labels used include dyes, antibodies or antibody fragments, metal chelating agents, spin labels, fluorophores, metal-containing moieties, radioactive moieties, actinic radiation excitable moieties, ligands, biotin, biotin analogs, redox-activators, isotopic labels. Moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron high density groups, magnetic groups, intercalating groups, chromophores; Energy transfer agents, quantum dot(s), fluorescent dyes, FRET reagents, and any combination thereof. The label is also any X ¹ as defined herein.

In one embodiment, such site-specific labeling of such proteins, polypeptides and/or peptides containing one or more pyrrolysine and/or PCL residues is pyrrolysine and/or PCL residue(s) with benzaldehyde, acetophenone. Or a reaction of a label derivatized with a benzophenone moiety. Labels used include dyes, antibodies or antibody fragments, metal chelating agents, spin labels, fluorophores, metal-containing moieties, radioactive moieties, actinic radiation excitable moieties, ligands, biotin, biotin analogs, redox-activators, isotopic labels. Moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron high density groups, magnetic groups, intercalating groups, chromophores; Energy transfer agents, quantum dot(s), fluorescent dyes, FRET reagents, and any combination thereof. The label is also any X ¹ as defined herein.

In a further aspect of such site-specific labeling provided herein, with a label derivatized with a residue that selectively reacts with pyrrolysine and/or PCL residue(s) with pyrrolysine and/or PCL residue(s). React to increase detection sensitivity. Specifically, these reactions form detectable moieties with different spectral properties than the reactants present before the reaction, thereby enhancing the ratio of signal to noise by minimizing the background signal associated with the detectable moiety signal. In certain embodiments of such site-specific labeling provided herein, the reaction of a pyrrolysine or PCL moiety(s) with a label derivatized with a benzaldehyde, acetophenone or benzophenone moiety is a reaction of a benzaldehyde, acetophenone or benzophenone moiety. Forms quinazoline-type residues with different spectral properties than those (Figures 22, 23 and 30). In certain embodiments of such site-specific labeling provided herein, the reaction of a pyrrolysine or PCL moiety(s) with a label derivatized with a benzaldehyde, acetophenone or benzophenone moiety is a reaction of a benzaldehyde, acetophenone or benzophenone moiety. Formed reduced quinazoline-type residues with different spectral properties than those (FIGS. 22, 23 and 30 ). In certain embodiments of such site-specific labeling provided herein, the reaction of a pyrrolysine or PCL moiety(s) with a label derivatized with a benzaldehyde, acetophenone or benzophenone moiety is a reaction of a benzaldehyde, acetophenone or benzophenone moiety. Fused ring moieties with different spectral properties than those (FIGS. 22, 23 and 30 ).

In certain embodiments, such labeled proteins, polypeptides and/or peptides are used for diagnosis and imaging, and in other embodiments, such labeled proteins, polypeptides and/or peptides are used in high-throughput screening. In other embodiments, such labeled proteins, polypeptides and/or peptides are used to assess transport properties, and in other embodiments, such labeled proteins, polypeptides and/or peptides are used in localization studies.

46 illustrates certain embodiments of such site specific labeling. 46A illustrates labeling with fluorescent moieties. 46B illustrates the reaction of PCL moieties with non-fluorescent moieties that become fluorescent upon reaction. Figure 46C illustrates the biotinylation of proteins. 46D illustrates labeling with radioactive moieties. Figure 46E illustrates labeling with 1-(2-amino-5-iodophenyl)ethanone, and Figure 46F illustrates labeling with 1-(2-amino-5-bromophenyl)ethanone, Both of these can be used to obtain phase information for a method of determining the X-ray crystal structure of a protein. 1-(2-amino-5-iodophenyl)ethanone can also be used for protein labeling with radioactive moieties. In Figure 46, the protein conjugate linkage formed by this labeling is a quinazoline-type residue, but in other embodiments the linkage is a reduced form of a quinazoline-type residue (Figures 22, 23 and 30). In other embodiments, the linkage is a fused ring moiety (FIGS. 22, 23 and 30 ).

In embodiments of such site-specific labeling of such proteins, polypeptides and/or peptides containing one or more pyrrolysine and/or PCL residue(s), precursors labeled with pyrrolysine or PCL residue(s) themselves It can be labeled with. 46G and 46H show, by way of example only, labeling by radioactive or stable isotopes from differently labeled precursors.

Another aspect of the invention provides the production of a protein that is homologous to any available protein but comprises one or more pyrrolysine or PCL residue(s). For example, in certain embodiments, a therapeutic protein comprising at least one pyrrolysine or PCL and homologous to at least one therapeutic protein is prepared. For example, in one aspect, the protein is homologous to a therapeutic or other protein, e.g., a cytokine, a growth factor, a growth factor receptor, an interferon, an interleukin, an inflammatory molecule, an oncogene product, a peptide hormone, a signal. Introducing molecule, steroid hormone receptor, transcription activator, transcription inhibitor, erythropoietin (EPO), fibroblast growth factor, fibroblast growth factor 21 (FGF21), leptin, insulin, human growth hormone, epithelial neutrophil activating peptide-78 , GROα/MGSA, GROβ, GROγ, MIP-1α, MIP-16, MCP-1, hepatocyte growth factor, insulin-like growth factor, leukemia inhibitory factor, oncostatin M, PD-ECSF, PDGF, pleiotropin, SCF, c-kit ligand, VEGF, G-CSF, IL-1, IL-2, IL-8, IGF-I, IGF-II, FGF (fibroblast growth factor), PDGF, TNF, TGF-α, TGF -ß, EGF (epidermal growth factor), KGF (keratinocyte growth factor), CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, hyalurin/CD44, Mos, Ras, Raf, Met ; p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and/or corticosterone. In another set of embodiments, the protein is homologous to a therapeutic or other protein, for example: alpha-1 anti-trypsin, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrial sodium Diuretic factor, atrial natriuretic polypeptide, atrial peptide, CXC chemokine, T39765, NAP-2, ENA-78, Gro-α, Gro-β, Gro-γ, IP-10, GCP-2, NAP-4, SDF- 1, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 alpha, monocyte Inflammatory protein-1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, C-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epidermal neutrophil activation peptide-78, GROα/MGSA, GROβ, GROγ, MIP-1α, MIP-16, MCP-1, epidermal growth factor (EGF), epidermal neutrophil activation peptide, interferon alpha (INF-α) Or interferon beta (INF-β), exfoliating toxin, factor IX, factor VII, factor VIII, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, G-CSF, GM-CSF, glucocerebrosidase, Gonadotropin, growth factor, growth factor receptor, hedgehog protein, hemoglobin, hepatocyte growth factor (HGF), hirudin, human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, Insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon, IFN-α, IFN-ß, IFN-γ, interleukin, IL-1, IL-2, IL-3, IL-4 , IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocytes Growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neuroturin, neutrophil inhibitor (NIF), oncostatin M, bone morphogenetic protein, oncogene product, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, human Growth hormone, pleiotropin, protein A, protein G, pyrogenic exotoxin A, B or C, relaxin, renin, SCF, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, soma Tomedin, somatostatin, somatotropin, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock Syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor growth factor (TGF), TGF-α, TGF-ß, tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor (TNFR) ), VLA-4 protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, Mos, Ras, Raf, Met; p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and/or corticosterone.

In one aspect, a composition herein comprises a protein comprising one or more pyrrolysine or PCL residue(s), such as any of the proteins described above, and a pharmaceutically acceptable excipient.

In certain embodiments, the protein is a therapeutic protein, in other embodiments, the therapeutic protein is erythropoietin (EPO), fibroblast growth factor 21 (FGF21), interferon alpha (INF-α) or interferon beta (INF-β). to be. In certain embodiments, the therapeutic protein is an antibody or antibody fragment including, but not limited to, a Fab. In certain embodiments, the therapeutic protein is produced as a fusion protein with a modified fusion partner. In certain embodiments, the therapeutic protein is a fusion with an Fc domain.

Homology to a protein or polypeptide can be inferred, for example, by setting default parameters to perform sequence alignment, eg BLASTN or BLASTP. For example, in one embodiment, the protein is at least about 50%, at least about 75%, at least about 80 with a known therapeutic protein (e.g., a protein present in Genebank or other available databases). %, at least about 90% or at least about 95% identical.

To demonstrate site specific labeling, PCL was incorporated into mEGF using the method provided herein (see Example 12), and the PCL residue was coupled to biotin via a polyether functionalized with an ABA residue (X3626 See -140, Example 40). Figure 47A shows the ESI mass spectrometric analysis of mEGF-Tyr10PCL conjugated with biotin (see Example 24). 47B shows Western blotting of mEGF-Tyr10PCL-ABA-biotin conjugates using horseradish peroxidase (HRP) conjugated goat anti-biotin antibody. Uncoupled mEGF-Tyr10PCL and fluorescein-conjugated mEGF-Tyr10PCL served as negative controls.

To further demonstrate site specific labeling, PCL was incorporated into mEGF using the method provided herein (see Example 12), and the PCL residue was coupled to fluorescein via a polyether functionalized with an ABA residue. Ringed (see X3757-48, Example 41). Figure 47C shows the ESI mass spectrometric analysis of mEGF-Tyr10PCL conjugated with fluorescein (see Example 25). In addition, PCL was incorporated into mEGF using the method provided herein (see Example 12), and the PCL residue was coupled to a disaccharide functionalized with an ABA residue (see 3793-050, Example 42). Figure 47D shows the ESI mass spectrometric analysis of mEGF-Tyr10PCL conjugated to a disaccharide (see Example 26). It is understood that the coupling chemistry used for attachment of the disaccharides can be readily applied to the coupling of other sugars and complex oligos and polysaccharides.

Coupling of an immunomodulatory agent to a pyrrolysine and/or pyrrolysine analogue protein incorporated in a protein, polypeptide and/or peptide and enhancement of immunogenicity by such coupling

In another aspect provided herein, proteins, polypeptides and/or peptides having at least one pyrrolysine and/or PCL incorporated therein are derivatized with at least one immunomodulatory agent. Using the methods provided herein, immune stimulating moieties can be readily coupled to proteins, polypeptides and/or peptides via pyrrolysine and/or PCL moiety(s). These immune stimulating moieties include one or more nitro groups, lipids, phospholipids, LPS-like molecules, adjuvants, adjuvant-like molecules, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T -Cellular epitope, keyhole limpet hemocyanin (KLH), immunogenic hapten, halogen, aryl group, heteroaryl group, cycloalkyl group or heterocycloalkyl group are included, but are not limited thereto. In certain embodiments, an immune modulator attached to pyrrolysine and/or PCL is used to stimulate an immune response to self-antigens in a manner similar to p-nitro-phenylalanine incorporated into the protein (Gruenewald J, Tsao ML, Perera R, Dong L, Niessen F, Wen BG, Kubitz DM, Smider VV, Ruf W, Nasoff M, Lerner RA, Schultz PG, Immunochemical termination of self-tolerance, Proc Natl Acad Sci US A. 2008 Aug 12; 105(32):11276-80]). In certain embodiments, self-antigens having an immunomodulatory agent attached to pyrrolysine and/or PCL are used as cancer vaccines. In another embodiment, an antigen having an immunomodulatory agent attached to pyrrolysine and/or PCL is used as a vaccine for infectious diseases. In other embodiments, antigens having an immunomodulatory agent attached to pyrrolysine and/or PCL are used as a vaccine against diseases involving the formation of amyloid, such as but not limited to Alzheimer's disease.

In one embodiment, a library of 2-aminobenzaldehyde compounds is generated, and after coupling such library to pyrrolysine or PCL incorporated into the antigen of interest, the coupled product is converted to a high titer antibody against the modified and unmodified antigens. Screening for the generation of immunostimulating moieties is generated. In such embodiments, the 2-aminobenzaldehyde moiety is one or more nitro groups, lipids, phospholipids, LPS-like molecules, adjuvants, adjuvant-like molecules, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists. , TLR8 agonist, T-cell epitope, keyhole limpet hemocyanin (KLH), immunogenic hapten, halogen, aryl group, heteroaryl group, cycloalkyl group or heterocycloalkyl group, including, but not limited to, various substituents. Is substituted.

In another embodiment, a library of 2-amino-acetophenone compounds is generated, the library is coupled to the pyrrolysine or PCL incorporated into the antigen of interest, and then the coupled product is transferred to the modified and unmodified antigens. A library of immune stimulating residues is generated by screening for the production of high titer antibodies against. In such embodiments, the 2-amino-acetophenone residue is one or more nitro groups, lipids, phospholipids, LPS-like molecules, adjuvants, adjuvant-like molecules, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9. Agonist, TLR8 agonist, T-cell epitope, keyhole limpet hemocyanin (KLH), immunogenic hapten, halogen, aryl group, heteroaryl group, cycloalkyl group or heterocycloalkyl group including, but not limited to, a variety of Substituted with a substituent.

In another embodiment, a library of 2-amino-5-nitro-benzophenone compounds is generated, and after coupling such library to pyrrolysine or PCL incorporated into the antigen of interest, the coupled product is modified antigen and A library of immune stimulating residues is generated by screening for the production of high titer antibodies against unmodified antigens. In this embodiment, the 2-amino-5-nitro-benzophenone moiety is one or more nitro groups, lipids, phospholipids, LPS-like molecules, adjuvants, adjuvant-like molecules, TLR2 agonists, TLR4 agonists, TLR7. Agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, keyhole limpet hemocyanin (KLH), immunogenic haptens, halogens, aryl groups, heteroaryl groups, cycloalkyl groups, or heterocycloalkyl groups. Not) is substituted with various substituents.

To demonstrate this aspect, PCL was incorporated into mTNF-α and mEGF using the method provided herein (see Examples 11 and 12), and the PCL residue was coupled to a hapten containing at least one nitrophenyl group. (See Examples 27 and 28). Figures 48A and 48B demonstrate the attachment of mono-nitrophenyl hapten (see 3793-001, Examples 38-8) at the site of PCL incorporation of mTNF-Gln21PCL (Figure 48A) and mEGF-Tyr10PCL (Figure 48B). Figures 48C and 48D show the attachment of di-nitrophenyl hapten (TU3627-088, Example 38-7) at the site of PCL incorporation of mTNF-α (Figure 48C) and mEGF (Figure 48D).

Figure 49A demonstrates the attachment of a TLR7 agonist (see X3678-114, Example 38-3) at the site of PCL incorporation of mEGF-Tyr10PCL (see Example 29). Figure 49B demonstrates the adhesion of phospholipids (see TU3627-092, Example 43-1) at the site of PCL incorporation of mEGF-Tyr10PCL (see Example 31).

(여기서, X는 시클로헥실-알라닌임)이다. exPADRE 펩티드의 서열 (서열 29)은

(여기서, X는 시클로헥실-알라닌임)이다. 특정 실시양태에서, 공지된 면역원성 펩티드 에피토프는 항원과 커플링되어 상기 항원의 면역원성을 증진시킬 것이다. 특정 실시양태에서, 항원 유래의 펩티드는 면역원성 운반 단백질과 커플링될 것이다. 특정 실시양태에서, 면역원성 운반 단백질은 KLH이다. PADRE 펩티드의 부착에 이용되는 커플링 화학은 PCL 및 피롤리신에 대한 다른 펩티드의 커플링에도 쉽게 적용될 수 있음이 이해된다.Figures 50-51 show PADRE peptides against mTNF-Gln21PCL or mEGF-Tyr10PCL: PX2-PADRE (MW = 1585) (see 3465-143; Example 38-11) and BHA-exPADRE (MW = 2060) (3647-104 See; Examples 38-10) demonstrate conjugation. 50A and 50B show MALDI-TOF mass spectrometric analysis of mTNF-Gln21PCL conjugation reaction with PX2-PADRE at two different pH values (see Example 30), and FIG.50C is mTNF-Gln21PCL conjugation with BHA-exPADRE. The ESI mass spectrometric analysis of the reaction is shown (see Example 30). Figure 51 shows the coupling of BHA-exPADRE to mEGF-Tyr10PCL (see Example 30). The sequence of the PADRE peptide (SEQ ID NO: 28) is

(Wherein X is cyclohexyl-alanine). The sequence of the exPADRE peptide (SEQ ID NO: 29) is

(Wherein X is cyclohexyl-alanine). In certain embodiments, a known immunogenic peptide epitope will be coupled with an antigen to enhance the immunogenicity of the antigen. In certain embodiments, the antigen-derived peptide will be coupled with an immunogenic carrier protein. In certain embodiments, the immunogenic carrier protein is KLH. It is understood that the coupling chemistry used for attachment of the PADRE peptide can be readily applied to the coupling of PCL and other peptides to pyrrolysine.

Immuno- PCR : Coupling of DNA and other oligonucleotides to pyrrolysine and/or pyrrolysine analogs incorporated into proteins, polypeptides and/or peptides

In another aspect provided herein, proteins, polypeptides and/or peptides having at least one pyrrolysine and/or PCL incorporated therein are derivatized to DNA using the methods provided herein. DNA is modified to have a terminal 2-amino-benzaldehyde, 2-amino-acetophenone or 2-amino-5-nitro-benzophenone residue that reacts with pyrrolysine and/or PCL incorporated in the protein, polypeptide and/or peptide. And the DNA is coupled to the protein, polypeptide and/or peptide via a quinazoline-type linkage, a reduced quinazoline linkage, or a fused ring linkage (see FIGS. 22, 23 and 30).

(여기서, *는 포스포티오에이트 연결부를 나타냄)이다. BG2의 서열 (서열 31)은

(여기서, *는 포스포티오에이트 연결부를 나타냄)이다. 특정 실시양태에서, 커플링될 올리고뉴클레오티드는 데옥시리보핵산, 리보핵산, 펩티드 핵산 또는 기타 변형된 올리고뉴클레오티드일 것이다. 처음에는 피롤리신 또는 PCL에 단일 가닥 올리고뉴클레오티드로서 커플링시키고, 이중 가닥 DNA, RNA, PNA 또는 하이브리드 중합체는 상보적 가닥과의 혼성화로 쉽게 제조될 수 있다. CpG 올리고뉴클레오티드의 부착에 사용된 커플링 화학은 다른 올리고뉴클레오티드의 커플링에 쉽게 적용될 수 있다는 것이 이해된다.In order to demonstrate the attachment of DNA to PCL, PCL was incorporated into mTNF-α and mEGF using the method provided herein (see Examples 11 and 12), and the PCL residue was CpG oligonucleotide, which is also an immune stimulating residue. Coupled to (see Example 32). BHA-BG1 (see 3647-057, Example 38-12) and BHA-BG2 (see 3597-167, Example 38-14) were used to attach CpG to mTNF-Gln21PCL and mEGF-Tyr10PCL at the site of PCL incorporation. It was a CpG reagent. The coupling of BHA-BG1 (7.4 kDa) and BHA-BG2 (7.4 kDa) to mTNF-Gln21PCL (19.3 kDa) was confirmed by a gel shift assay (Figure 52A), and BHA- to mEGF-Tyr10PCL (7.2 kDa). The coupling of BG2 (7.4 kDa) (Fig. 52B) was similarly confirmed. The sequence of BG1 (SEQ ID NO: 30) is

(Here, * indicates a phosphorothioate linkage). The sequence of BG2 (SEQ ID NO: 31) is

(Here, * indicates a phosphorothioate linkage). In certain embodiments, the oligonucleotide to be coupled will be a deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, or other modified oligonucleotide. Initially coupled to pyrrolysine or PCL as single-stranded oligonucleotides, double-stranded DNA, RNA, PNA or hybrid polymers can be readily prepared by hybridization with complementary strands. It is understood that the coupling chemistry used for the attachment of CpG oligonucleotides can be readily applied to the coupling of other oligonucleotides.

In certain embodiments, the protein, polypeptide and/or peptide is an antibody, and immuno-PCR analysis is performed using such an antibody to which DNA is linked. Immuno-PCR is performed by ligating DNA to an antibody using the methods described herein, followed by binding of the DNA-linked antibody to a target antigen. After binding, DNA is amplified by DNA amplification technology, and the resulting amplified product is analyzed by electrophoresis technology, microplate method, or real-time PCR.

Site specific and orientation attachment

In another aspect provided herein, site specific incorporation of one or more pyrrolysines and/or PCLs into proteins, polypeptides and/or peptides upon attachment to a support surface, such proteins, polypeptides and/or Controls the orientation of the peptide. This attachment is due to the reaction of a surface derivatized with a pyrrolysine or PCL moiety with a benzaldehyde moiety, acetophenone moiety, benzophenone moiety or a combination thereof, whereby a quinazoline moiety linkage is formed and the protein is oriented on the surface. . By incorporating pyrrolysine and/or PCL at specific positions in the protein, the orientation of the protein on the surface is controlled. This control over the direction of protein attachment is used as a component of a protein engineering tool kit for evaluating protein properties. Figure 53 illustrates an embodiment of such a site-specific orientation attachment, wherein the surface is derivatized with 20 amino-acetophenone residues that react with PCL residues incorporated into the protein, allowing the protein to be described above through a quinazoline-type linkage. Attach it to the surface. In Figure 53 the protein conjugate linkage formed is a quinazoline-type moiety, but in other embodiments the linkage is a reduced form of a quinazoline-type moiety (see FIGS. 22, 23 and 30). In other embodiments, the linkage is a fused ring moiety (see FIGS. 22, 23 and 30).

Non-limiting examples of supports include solid and semi-solid matrices such as aerogels and hydrogels, resins, beads, biochips (e.g., thin films coated with biochips), microfluidic chips, silicon chips, multi-well plates (also (Also referred to as microtiter plates or microplates), membranes, cells, conductive and non-conductive metals, glass (eg, microscope slides), and magnetic supports. Other non-limiting examples of solid supports used in the methods and compositions described herein include silica gel, polymer membranes, particles, derivatized plastic films, derivatized glass, derivatized silica, glass beads, cotton, plastic beads, Alumina gels, polysaccharides such as sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, Mannan, inulin, nitrocellulose, diazocellulose, polyvinyl chloride, polypropylene, polyethylene (e.g., poly(ethylene glycol)), nylon, latex beads, magnetic beads, paramagnetic beads, superparamagnetic beads, starch, etc. do. In certain embodiments, the support used in the methods and compositions described herein is a support used for surface analysis, such as a surface acoustic wave device or an apparatus using vanishing wave analysis, such as surface plasmon resonance analysis.

The surface of the solid support used for attachment of proteins, polypeptides and/or peptides having pyrrolysine and/or pyrrolysine analogs incorporated therein is hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyanide. It has reactive functional groups including, but not limited to, furnace, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide and sulfoxide. Such a functional group comprises a 2-amino-benzaldehyde residue, a 2-amino-acetophenone residue, and/or a 2-amino-5-nitro-benzophenone residue that reacts with pyrrolysine or a pyrrolysine analog to form a quinazoline residue. Used for covalent attachment. In certain embodiments, such 2-amino-benzaldehyde moieties, 2-amino-acetophenone moieties and 2-amino-5-nitro-benzophenone moieties are solid support reactive functional groups such as but not limited to hydroxyl, car Coupled to a solid support by reaction with boxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide and sulfoxide. It is part of the polymer linker.

In other embodiments, the surface of the solid support has streptavidin or avidin attached thereto, and is used for attachment of proteins, polypeptides and/or peptides having pyrrolysine and/or pyrrolysine analogs incorporated therein, and , Wherein the pyrrolysine and/or pyrrolysine analog is used to site-specifically attach biotin to the protein, polypeptide and/or peptide.

Other supports used in the methods and compositions described herein include resins used for peptide synthesis, such as polystyrene, PAM-resin, POLYHIPE™ resins, polyamide resins, poly(ethylene, by way of example only). Glycol) implanted polystyrene resin, polydimethyl-acrylamide resin, and PEGA beads.

In certain embodiments, proteins, polypeptides and/or peptides with pyrrolysine and/or PCL incorporated therein are deposited on a solid support in an array format. In certain embodiments, such deposition is carried out by direct surface contact between the support surface and a delivery mechanism, such as a pin or capillary, or by ink jet technology using piezoelectric and other forms of propulsion to move liquid from a small nozzle to a solid surface. . For contact printing, robotic control systems and multiplexed printheads allow automated microarray fabrication. In the case of non-contact deposition by piezoelectric propulsion technology, robotic systems also allow automatic microarray fabrication using continuous or drop-on-demand devices.

Example

It should be understood that the embodiments described herein and the following examples are for illustrative purposes only, and that various modifications or changes thereto will be apparent to those skilled in the art and are included within the spirit and scope of the present application and the appended claims. All publications, patents and patent applications cited herein are incorporated herein by reference for the purposes as indicated.

Example 1 : Site-specific incorporation of biosynthetically generated PCL into protein using mammalian cells

This example provides a description of a gene construct that enables incorporation of PCL into a TAG coding site in a target protein when transfected with mammalian cells. While simultaneous incorporation at multiple sites is possible, all examples herein illustrate only PCL incorporation at a single site per protein molecule. This example also describes the general procedure for incorporating PCL into proteins using mammalian cells.

Construct:

Using PCR using primers designed based on the nucleotide sequences of pylS, pylT, pylB, pylC and pylD of metanosarcina majei Go1 (NC_003901, see below), full-length pylT from genomic DNA of metanosarcina majei Go1, And coding regions of pylS, pylT, pylB, pylC and pylD were amplified. The start codon of these genes was changed to ATG. pylT was cloned upstream of the CMV promoter into the pCMV vector. Transcription was under the control of the U6 promoter and formed the vector pCMVU6. pylS, pylB, pylC and pylD were similarly cloned into pCMVU6. The transcription of pylS, pylB, pylC and pylD was under the control of the CMV promoter (Fig. 4A).

The coding regions of target genes human retinol binding protein (hRBP4), human and mouse erythropoietin (EPO) and mIgG1 Fc (mFc) were cloned into a pRS vector under CMV promoter control by having a His tag at the C-terminus (Fig. 4B). Residues to be mutated to the TAG codon were selected based on solvent-exposure in the existing structural model as shown in Table 2. The TAG codon was introduced into the target gene by PCR.

Figure 4B shows the TAG mutant constructs of hRBP4, mEPO, hEPO and mIgG1. Mutation sites are listed in Table 2 and indicated by arrows in Figure 4B. For each construct, a His tag was attached to the C-terminus as a purification and detection tool. Only full-length proteins can carry His tags. pylT, pylS, pylB, pylC and pylD were cloned into pCMVU6 under CMV control (Fig. 4A). These were used in co-transfection studies.

Cell culture and transfection:

HEK293F cells were grown as a suspension in 293 Freestyle expression medium at 37° C. under 5% CO _2. One day before transfection, cells ^{were split at 0.7×10 6} cells/mL. Plasmid DNA was prepared with a Qiagen Maxi plasmid preparation kit. Transfection was carried out by the PEI method. PEI was mixed with plasmid DNA in Opti-MEM at a ratio of 2:1, DNA complex was added to HEK293F cells with 1 μg plasmid DNA per 1 mL of cell culture, and the cells were added to 5 mM Cyc or D- Incubated in the presence of ornithine. When cells were co-transfected with several plasmids, the ratio of the total amount of PEI:plasmid DNA was always 2:1.

Protein purification and analysis:

After 4 days of transfection, the cell culture was centrifuged at 2000 g for 20 minutes, and a medium was collected for purification of the His-tagged protein. The medium was loaded onto a Ni-NTA column previously equilibrated with 20 mM Tris-HCl (7.5) and 150 mM NaCl containing 10 mM imidazole. The column was washed with the same buffer and eluted with an elution buffer (20 mM Tris-HCl 7.5, 150 mM NaCl, 300 mM imidazole). The eluted protein was assayed by Bradford method and SDS-PAGE. In some cases, media and purified proteins were analyzed by Western blotting using His tags or antibodies against the protein.

The sequence of the pyl gene cloned from the genomic DNA of metanosarcina majei is shown below:

Example 2: The above portion to the mammal of an animal using the cells generated by biosynthetic PCL hRBP4 specific incorporation

This example demonstrates the site-specific incorporation of PCL at the TAG coding site in the model protein human RBP4 (human retinol binding protein 4). hRBP4 is a secreted protein, its structure has been characterized, and the solvent exposed residues in hRBP4 are defined.

hRBP4 was cloned into a pRS vector to have a Flag-His tag at the C-terminus (Fig. 4A). HRBP4 was sufficiently expressed in HEK293F cells using transient transfection. TAG codons were introduced at 9 positions in separate hRBP4 constructs as shown in Table 2 and the sequence below. Note that individual TAG codons were introduced at the codons of the underlined amino acid residues.

Tyr108 and Phe54 are the retinol binding sites of hRBP4, but all other TAG mutations are present at the protein residues exposed to the solvent. When expressed in mammalian cells, termination of translation at these TAG sites results in a truncated protein without a C-terminal His tag. Thus, these truncated proteins will not be recognized by anti-His antibodies. Read-through translation will produce a full-length product with a His tag. Thus, detection with an anti-His antibody serves as an indicator of transcriptional progression activity.

hRBP4 Manifestations of:

pRSRBP was co-transfected with pCMVpyS, pCMVpyB, pCMVpyC and pCMVpyD into HEK293F cells, and cells were grown in the presence of 5 mM D-ornithine as described in Example 1. Subsequently, using Western blotting and anti-His antibody, hRBP4 with His tag was analyzed, and since truncated hRBP4 does not contain His tag, only full-length hRBP4 having PCL incorporated in the TAG codon was detected by Western blotting. Became. Thus, the presence of full-length hRBP4 demonstrates PCL incorporation. As shown in Fig. 6A, the anti-His antibody detected a band at 26 kDa, which is the expected size for full-length hRBP4, indicating that PCL was incorporated into the mutated TAG codon. PCL was incorporated at different rates in the 9 hRBP4 constructs (Table 2), higher rates in #2, #6 and #9 mutant constructs and lower in #7 and #8 mutant constructs. Protein yields for #2, #6 and #9 were 4 to 8 mg/L, and for wild-type hRBP4 it was about 20% of the yield. These proteins were purified and subjected to mass spectrometry (MS) analysis. The mass obtained for the protein was consistent with that expected for the incorporation of PCL, and parallel MS analysis confirmed the incorporation of PCL at the expected site (FIGS. 6A to 11 ).

6B shows the SDS-PAGE of hRBP4 generated in HEK293F cells in which PCL was incorporated into hRBP4, and FIG. 6C shows its mass spectrum. hRBP4 was purified from the medium of co-transfected HEK293F cells in the absence of D-ornithine (A, lane 1) or in the presence of D-ornithine (A, lane 2) and analyzed by SDS-PAGE. Arrows indicate full length hRBP4. The purified protein was analyzed by mass spectrometry (B): The observed mass was 23166.0 Da, which was similar to the expected mass of 23168 Da.

hRBP4 Specifically incorporated in the PCL Mass spectrometry detection of:

(여기서, 잔기 F*는 PCL의 질량과 일치하는 질량을 가짐)에 배정될 수 있고, 이는 잔기 122의 원하는 TAG 부위에서의 PCL의 부위 특이적 혼입을 확인시켜 준다. 펩티드

에 대한 배정은 하기 기재한다:500 mL cultures of HEK293F cells transfected with DNA for the pylS, pylT, pylC and pylD genes, and hRBP4 mutant #6 (Table 2: hRBP4 Phe122PCL) were grown in medium supplemented with 5 mM D-ornithine. The medium was run twice on a Ni-NTA column to capture all full-length hRBP4 proteins. The combined elution fractions contained 4.3 mg of total protein. The observed mass of the protein was 23166.8 Da (estimated 23168 Da), which was consistent with the incorporation of a single PCL residue (data not shown). Sample aliquots were trypsin digested and LC-MS analysis was performed. The MS/MS spectrum of Figure 7 shows the peptide

(Where residue F* has a mass that matches the mass of PCL), which confirms the site-specific incorporation of PCL at the desired TAG site of residue 122. Peptide

Assignments to are listed below:

에 대한 배정 (F* = PCL)m/z at 647.86

For (F* = PCL)

Monoisotopic mass of neutral peptide Mr (calculated value): 1273.6819

Variable transformation:

F7: PCL at F (F)

Ion Score: 60 Expected: 0.0013

Match (bold): 22/74 fragment ions (using 32 strongest peaks)

의 2+ 이온의 TIC 및 EIC)을 보여주며, 표적 Phe122TAG 부위에서의 PCL의 혼입을 나타낸다. 도 9는 인간 RBP4 Phe122PCL의 트립신 소화물의 질량 분광측정 분석을 보여준다. 질량 스펙트럼은 m/z 425.57 (3+) 및 637.85 (2+) 각각에서

의 3+ 및 2+ 전구체를 보여주고 (F* = PCL), 이것은 표적 Phe122TAG 부위에서의 PCL의 혼입을 추가로 나타낸다.Figure 8 is a mass spectrometric analysis of the trypsin digest of human RBP4 Phe122PCL (

TIC and EIC of the 2+ ion of Phe122TAG), indicating incorporation of PCL at the target Phe122TAG site. Figure 9 shows the mass spectrometric analysis of the trypsin digest of human RBP4 Phe122PCL. Mass spectra at m/z 425.57 (3+) and 637.85 (2+) respectively

The 3+ and 2+ precursors of are shown (F* = PCL), which further indicates incorporation of PCL at the target Phe122TAG site.

Example 3 : Detection of biosynthetically generated PCL in lysates from mammalian cells

This example demonstrates that PCL is produced biosynthetically in mammalian cells and is detectable as a free amino acid in the lysate of such cells. Thus, this example suggests that PCL is incorporated like pyrrolysine and other 20 naturally occurring amino acids, and that PCL incorporation is not the result of post-translational protein modification.

cell Lysate medium PCL Detection of

60 mL cultures of HEK293F cells were grown with the pylT, pylS, pylB, pylC and pylD genes in the presence of 5 mM D-ornithine or without D-ornithine. The cells were harvested and lysed by sonication in 250 μl of double-deionized water. Cell debris was pelleted by centrifugation. The protein was precipitated by adding cold methanol to a final concentration of 80% and removed by centrifugation. The soluble lysate was then concentrated with a speedbag and analyzed by high pressure liquid chromatography in combination with mass spectrometry to confirm the presence of PCL.

The dried samples were first reconstituted in 100 μl of 98/2 mobile phase A/mobile phase B (mobile phase A: water containing 0.1% formic acid; mobile phase B: acetonitrile containing 0.1% formic acid). Then, the sample was diluted 20 times, and 2 μl was injected into the HPLC. Separation of analytes was performed on an Agilent zorbax 300SB_C18 reverse phase HPLC column using the following solution gradient: 0 min 2% B; 5 min 2% B; 60 min 100% B; Flow rate: 0.25 mL/min. Comparison of HPLC traces (Fig.10A) is present in the lysate of cells transfected with biosynthetic genes pylB, pylC and pylD and grown in the presence of D-ornithine (lower EIC (extracted ion chromatogram) trace), but It shows the peak at 4.13 min (marked with an asterisk) that is not present in the lysate of cells grown without D-ornithine (top EIC trace). The full scanning mass spectrum of the 4.13 minute HPLC peak (Figure 10C) shows the mass of 242.14943 Da, which is consistent with the theoretical mass for PCL, the demethylated version of PYL (242.14992 Da). Lysine (HPLC peak at 1.44 min) is equally abundant in both samples (Figure 10B), and the full mass spectrum of lysine (Figure 10D) serves as an internal calibration and illustrates the accuracy of the method. This analysis indicates that PCL is produced as a detectable free amino acid from D-ornithine in cell lysate.

Example 4 : Incorporation test using putative PCL and pyrrolysine precursors, synthetic pyrrolysine analogs, and different combinations of biosynthetic genes

This example demonstrates that D-ornithine is a preferred precursor of PCL biosynthesis as measured by site specific incorporation into human RBP4. In addition, in this Example, the site-specific incorporation of a specific pyrrolysine analog, such as CYC, at the TAG coding site in the model protein human RBP4 and human retinol binding protein 4 using mammalian cells is exemplified. This example also provides insight into the substrate specificity of the pylS tRNA synthetase.

N-ε- Cyclopentyloxycarbonyl -L-lysine ( CYC ) Mixed

DNA constructs for 9 hRBP4 TAG mutants (Table 3) were individually co-transfected into HEK293F cells with pylT/pylS DNA as described in Example 2, and 4 mM N-ε-, a pyrrolysine analogue. Incubated without or in the presence of cyclopentyloxycarbonyl-L-lysine (CYC). Culture medium was harvested and analyzed by Western blotting using anti-His and anti-hRBP4 antibodies (Fig. 11A). Western blotting with anti-His antibody revealed a protein of 24 kDa for 6 out of 9 hRBP4 TAG mutant constructs. The size of the protein corresponds to the size of wild-type hRBP4, indicating the production of a full-length protein through transcriptional progression translation. Transcription progression activity was different for each hRBP4 mutant construct (Table 3) and was CYC and pylS dependent. In particular, the full-length hRBP4 protein was not detected in mutants #1, 5 and 7. High-yield full-length proteins were observed in mutants #2, 6 and 9, which were similar to the results shown in the case of PCL incorporation (Example 2, Fig. 6A).

의 MS/MS 단편화는 다음과 같다:The expressed protein containing CYC was purified from the medium using Ni-NTA chromatography and analyzed by SDS-PAGE followed by Coomassie blue staining. 11B shows SDS-PAGE analysis of purified hRBP4 TAG mutant #2. The purified formulation showed a single protein band of size 24 kDa. The mass spectrum of the purified protein was consistent with the single site incorporation of CYC (Fig. 11C). The expected molecular weight of single site CYC incorporation instead of Phe62 was (23114.6 Da (wild type)-165.2 Da (Phe) + 258.3 Da (CYC)) 23208.7 Da, and the observed value was 23182.0 Da. The observed mass suggests that the N-terminal Q moiety is cyclized with pyrrolidone carboxylic acid resulting in 18 Da missing and 6 Da missing due to the presence of 3 intact disulfide bonds (23208.7 Da-6 Da- 18 Da = 23184.7 Da). Parallel MS analysis of the purified protein demonstrated that CYC was incorporated into the designated TAG site in the hRBP4 construct (FIG. 12 ). The protein yield from transient transfection was estimated to be approximately 5 mg/L.

The MS/MS fragmentation of is as follows:

Monoisotopic mass of neutral peptide Mr (calculated value): 3248.5646

Variable transformation:

F7: Cyc at F (F)

Q9: Deamidation (NQ)

N11: deamidation (NQ)

M24: oxidation (M) (neutral loss 0.0000) (shown in table), 63.9983

Ion Score: 113 Expected: 4.6e-009

Match (bold): 110/498 fragment ions (using 156 strongest peaks)

Putative PCL And incorporation test using a pyrrolysine precursor

The production of the full-length hRBP4 Phe122PCL protein (mutant #6) was determined by the putative PCL precursors D-ornithine, D-proline, D-arginine, D-glutamic acid, 4-hydroxyl-D-proline and 2-pyrrolidone-. It was determined in the presence of 5-carboxylic acid (Figs. 13A and B, Fig. 14A). pRSRBP was co-transfected with HEK293F cells with pCMVpyS, pCMVpyB, pCMVpyC and pCMVpyD, and the cells were grown in the presence of 5 mM of the putative precursor as described in Example 2 (Fig. 14A). Subsequently, full-length hRBP4 with His tag attached and PCL incorporated was analyzed by Western blotting and anti-His antibody and SDS-PAGE. As shown in Fig. 13B, only D-ornithine produced a measurable protein band on the SDS-PAGE of the Ni-NTA purified sample. Detection by Western blotting of the unpurified sample (FIG. 13A) showed that the full-length hRBP4 protein was formed only at low levels in the presence of all other precursors, indicating that D-ornithine is the most efficient precursor for biosynthesis and incorporation of PCL. Is clearly indicated. From Western blotting and SDS-PAGE analysis, biosynthetic production of PCL from 5 mM D-ornithine (lane 2) and subsequent protein incorporation was a known PylS substrate added at 5 mM to the medium of cells transfected with pCMVpyS. It is believed to be more efficient than the incorporation of CYC (lane 9). This example demonstrates that D-ornithine is a preferred precursor for the biosynthesis of PCL.

Incorporation test using synthetic pyrrolysine analogs

Contrary to the above experiment, the production of full-length hRBP4 Phe122PCL protein (mutant #6) was also determined in the presence of a series of synthetic pyrrolysine analogs designed to have acetyl residues for chemical derivatization after protein incorporation (Fig. 14B). . Synthetic analogs were prepared as described in Example 19. pRSRBP was co-transfected into HEK293F cells with pCMVpyS providing one gene copy of pylT tRNA and pylS tRNA synthetase. Depending on solubility, synthetic analogues were added to the medium at a final concentration of 2 or 5 mM. Subsequently, detection of full-length hRBP4 with His tag attached and PCL incorporated was analyzed by Western blotting and anti-His antibody and SDS-PAGE. 13B, only CYC (lane 9) produced a measurable protein band in the analysis of the Ni-NTA purified sample. Western blotting analysis of the crude sample (FIG. 13A) suggests that TU3000-016 (lane 15) is also a usable substrate for pylS tRNA synthetase. However, the incorporation efficiency is low, and the compound is unstable when it is determined by nuclear magnetic resonance spectroscopy that the different batches (TU2982-150) are decomposed and much less incorporated into hRBP4 (lane 10). Consistent with previous published reports, this example demonstrates that pyrrolysine analogs characterized by the sp2 carbon at the point of attachment of the ring moiety are not acceptable as substrates for pylS tRNA synthetase.

Incorporation test using various combinations of biosynthetic genes

The production of full-length hRBP4 Phe122PCL was also tested by co-transfection using cell cultures containing 5 mM D-ornithine and several combinations of biosynthetic genes pylB, pylC and pylD (Fig. 13C). All cultures were co-transfected with pCMVpyS providing one gene copy of pylT tRNA and pylS tRNA synthetase. Full-length hRBP4 protein is detected by Westin blotting with anti-His tag antibodies only when both pylC and pylD are co-transfected. Although pylB is believed to be necessary for PYL biosynthesis, it is not essential for PCL biosynthesis and subsequent incorporation. These observations are further confirmed by the generation of full-length PCL mutants of mIgG1 Fc domain protein (Example 5) and mouse and human EPO (Example 6). All three examples illustrate that only genes pylC and pylD are essential for PCL biosynthesis and protein incorporation.

Example 5 : Site-specific incorporation of biosynthetically generated PCL into the Fc domain of mouse IgG1 using mammalian cells

This example demonstrates that site-specific incorporation of biosynthetically generated PCL in mammalian cells using the methods provided herein is a general procedure and is not limited to the protein hRBP4.

mIgG1 Fc Manifestation of

Four types of TAG mutants were generated in the Fc domain of mouse IgG1 K333 (mutant #1), K336 (mutant #2), T394 (mutant #3) and L426 (mutant #4) (Table 2) And Figure 17A). pRSFc#1-4 (Table 2) was co-transfected into HEK293F cells with pCMVpyT, pCMVpyS, pCMVpyC and pCMVpyD, and the cells were grown in the presence of 5 mM D-ornithine (pCMVpyB was not added). The His-tagged Fc domain protein was purified from the medium using Ni-NTA chromatography and analyzed by SDS-PAGE. For each construct, the size of the protein bands in the gel was consistent with their expected size for the full-length protein (Fig. 17A). Only the full length protein was recovered because the expression construct was characterized by a C-terminal His6 tag for purification. As shown in Fig. 17A, the expression of mutant #1 depends on whether or not D-ornithine is added to the growth medium. The expression levels of all four mutants were similar. When the Fc domain is produced in HEK293F cells, mass spectrometric analysis was not performed because it is glycosylated.

Example 6 : Site-specific incorporation of biosynthetically generated PCL into erythropoietin (EPO) using mammalian cells

In this example, PCL was site-specifically incorporated into erythropoietin, which further demonstrates that the method provided herein is a general procedure for site-specific incorporation of biosynthetically generated PCL into proteins in mammalian cells.

Mouse erythropoietin ( EPO ) Expression

PCL incorporation into the EPO mutant protein was performed in HEK293F cells. TAG mutations were introduced into 11 surface-exposed Lys or Arg residues away from the EPO receptor binding interface. The incorporation sites are shown below and are shown in Table 2. Mouse EPO protein was expressed as described in Example 1, except that the pylB gene was not used: pRSEPO#1-11 (Table 2) was co-transfected into HEK293F cells with pCMVpyT, pCMVpyS, pCMVpyC and pCMVpyD. Infected and cells were grown in the presence of 5 mM D-ornithine. EPO with His tag was purified from the medium using Ni-NTA chromatography and analyzed by SDS-PAGE. The EPO construct contains a C-terminal His-tag. Thus, only proteins in which PCL has been successfully incorporated will produce full-length proteins and will be purified by Ni-NTA chromatography, thus producing a detectable band on SDS-PAGE. SDS-PAGE of full-length mouse EPO showed successful incorporation of PCL in all 11 mutants (Fig. 17B). For each construct, the size of the protein bands in the gel was consistent with their expected size for the full-length protein. In the case of mutant #1, it is exemplified that the formation of the full-length protein depends on the presence or absence of D-ornithine. The expression levels of all 11 mutant proteins were similar, ranging from 10% to 20% of the wild-type protein expressed at about 40 mg/L (FIG. 17B ). Mass spectrometric analysis was not performed because EPO is glycosylated in HEK293F cells. The sequences of the mature human (SEQ ID NO: 19) and mouse (SEQ ID NO: 20) EPO proteins are shown below, the PCL incorporation site is indicated in bold and the glycosylation site is underlined. Mutant numbering begins at the N-terminus (see also Table 2).

Example 7 : When using Escherichia coli cells, plasmid for incorporating biosynthetically generated PCL into protein

This Example relates to a plasmid capable of incorporating PCL into a TAG coding site in a target protein expressed from a second plasmid co-transformed with the same Escherichia coli cell when transformed with Escherichia coli cells. Provide a description.

pAra - pylSTBCD build

pylS PCR 생성물을 KpnI 및 SbfI로 소화시키고 KpnI 및 PstI 부위 사이에서 pSUP-pylT로 라이게이션하여 pAra-pylST를 생성하였다. pylB, pylC 및 pylD 생성물을 각각의 효소로 절단하고, NdeI 및 KpnI로 제조된 플라스미드 주쇄로 라이게이션하였다. 이러한 4가지 라이게이션의 플라스미드 생성물을 서열분석 및 진단 PCR로 확인한 후, 전체 카세트를 프라이머로 증폭시켜서 pylD-pylB-pylC 카세트의 양 말단에 KpnI 부위를 부가하였다. 이것을 KpnI로 소화시키고 KpnI 부위에서 pAra-pylST에 라이게이션하여 최종 pAra-pylSTBCD를 생성하였다 (도 5). 상기 최종 플라스미드는 pylD, pylB, pylC 및 pylS를 함유하고, 이것 각각은 별개의 아라비노스-유도성 및 T7 하이브리드 프로모터의 제어를 받고 각각마다 rrnB 종결자 하류 (pMH4 내용물과 동일함) 및 proK 프로모터하의 pylT 유전자 (단일 tRNA 카피를 갖는 pSUP/pSUPAR 내용물과 유사함)를 갖는다 ([Cellitti et al., "In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy", J Am Chem Soc. 2008 Jul 23; 130(29):9268-81]).this. A plasmid for incorporating PCL in E. coli cells is shown in Fig. 5, and was constructed as follows: a cassette encoding pylT under the proK promoter was synthesized by amplification of the 3'sequence of the promoter, tRNA, and pSUP (using overlapping primers). I did. Subsequently, the three fragments were mixed with terminal primers encoding restriction enzyme sites for ApaLI and XhoI to synthesize the entire insert. After digestion with ApaLI and XhoI, the cassette was ligated to the pSUP backbone prepared with the same enzyme to produce pSUP-pylT. M. Coding sequences for pylS, pylB, pylC and pylD from MaJ were amplified from the appropriate pCMVU6 plasmid prepared in advance (Example 1) and inserted into pMH4 without tags (Lesley SA, Kuhn P, Godzik A, Deacon AM , Mathews I, Kreusch A, Spraggon G, Klock HE, McMullan D, Shin T, Vincent J, Robb A, Brinen LS, Miller MD, McPhillips TM, Miller MA, Scheibe D, Canaves JM, Guda C, Jaroszewski L, Selby TL, Elsliger MA, Wooley J, Taylor SS, Hodgson KO, Wilson IA, Schultz PG, and Stevens RC, "Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline", Proc Natl Acad Sci USA 2002; 99: 11664-11669). Then, each entire promoter-CDS-terminator was amplified with primers having various restriction enzyme sites.

The pylS PCR product was digested with KpnI and SbfI and ligated with pSUP-pylT between the KpnI and PstI sites to generate pAra-pylST. The pylB, pylC, and pylD products were digested with respective enzymes and ligated into the plasmid backbone prepared with NdeI and KpnI. After confirming the plasmid products of these four ligations by sequencing and diagnostic PCR, the entire cassette was amplified with primers to add KpnI sites to both ends of the pylD-pylB-pylC cassette. This was digested with KpnI and ligated to pAra-pylST at the KpnI site to produce the final pAra-pylSTBCD (Fig. 5). The final plasmid contains pylD, pylB, pylC and pylS, each of which is under the control of a separate arabinose-inducible and T7 hybrid promoter, each downstream of the rrnB terminator (same as the pMH4 contents) and under the proK promoter. It has a pylT gene (similar to the pSUP/pSUPAR content with a single tRNA copy) ([Cellitti et al., "In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance). spectroscopy", J Am Chem Soc. 2008 Jul 23; 130(29):9268-81]).

Example 8 : Site-specific incorporation of biosynthetically generated PCL into FAS-TE using Escherichia coli cells.

This example provides a description of the incorporation of PCL into the TAG-encoded site of human fatty acid synthetase (FAS-TE) expressed from a second plasmid co-transformed with the same Escherichia coli cells.

The thioesterase domain coding residues 2221 to 2502 of human fatty acid synthetase (FAS-TE) were expressed from a pMH4 vector with an N-terminal tag (MGDSKIHHHHHHENLYFQG) (SEQ ID NO: 21) (Lesley SA, Kuhn P, Godzik A , Deacon AM, Mathews I, Kreusch A, Spraggon G, Klock HE, McMullan D, Shin T, Vincent J, Robb A, Brinen LS, Miller MD, McPhillips TM, Miller MA, Scheibe D, Canaves JM, Guda C, Jaroszewski L, Selby TL, Elsliger MA, Wooley J, Taylor SS, Hodgson KO, Wilson IA, Schultz PG, and Stevens RC, "Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline", Proc Natl Acad Sci USA 2002; 99:11664-11669). The TAG codon was mutated using PIPE cloning (Klock, HE, Koesema EJ, Knuth MW, and Lesley, SA, "Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts", Proteins. 2008 May 1;71(2):982-94)), which is described in [Cellitti et al., "In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear. magnetic resonance spectroscopy", J Am Chem Soc. 2008 Jul 23;130(29):9268-81. The mutants tested for PCL incorporation were Leu2222TAG/Leu2223Ile and Y2454TAG (FIG. 18A ). HK100 cells were co-transformed with pMH4-FAS-TE plasmid and pAra-pylSTBCD and selected on LB+Kan+Cm plates. Liquid cultures were induced after growing at 37° C. in TB (Sigma)+Kan+Cm supplemented with 5 mM D-ornithine (Sigma or Nova Biochem). When OD ₅₉₅ = 0.8, cells were transferred to 30° C. and induced with 0.2% arabinose after 15-30 minutes. After induction, cells were grown for approximately 20 hours before harvesting by centrifugation. Cells were lysed by sonication in TBS+5% glycerol (pH 8). The soluble protein fraction was purified on Ni-NTA (Qiagen) chromatography according to the manufacturer's protocol. The yield was 46-80 mg/L in the case of FAS-TE Leu2222PCL/Leu2223Ile, and 155-186 mg/L in the case of FAS-TE Tyr2454PCL, 50 to 80% of the yield of the wild-type protein. The molecular size in SDS-PAGE and the molecular weight of the protein measured by mass spectrometry were consistent with the incorporation of PCL alone at the desired position (FIG. 18B).

The sequence of FAS-TE (SEQ ID NO: 22) with two PCL incorporation sites (bold and underlined) is shown below (for Leu2222PCL, residue Leu2223 is mutated to Ile2223):

Example 9 : Site-specific incorporation of biosynthetically generated PCL into FKBP12 using Escherichia coli cells.

This example describes the incorporation of PCL into the TAG-encoded site of FKBP12 expressed from a second plasmid co-transformed with the same Escherichia coli cells.

FKBP12 was expressed from a pET vector (Novagen) with an N-terminal tag (MGSSHHHHHHLEVLFQGP) (SEQ ID NO: 23). The TAG codon was introduced at the Ile90 position using PIPE cloning (Reference [Klock, HE, Koesema EJ, Knuth MW, Lesley, SA, "Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts" , Proteins. 2008 May 1;71(2):982-94). BL21(DE3) cells (Invitrogen) were co-transformed with pET-FKBP12 and pAra-pylSTBCD and selected on LB+Kan+Cm plates. Liquid cultures were grown at 37° C. in TB+Kan+Cm supplemented with 5 mM D-ornithine. When OD ₅₉₅ = 0.4, cells were transferred to 30° C. and induced 30 minutes later with 1 mM IPTG. Cells were grown for an additional 20 hours before harvesting. Cells were lysed and purified for FAS-TE. The purified protein was then dialyzed against TBS and digested with HRV-3C protease (2U per 1 mg FKBP12) at 4° C. for 48 hours to remove the His tag. The cut material was run on a Ni-NTA column, collected, concentrated, and passed through an S75 size exclusion column (GE Healthcare) for further purification. The final protein was further concentrated to 15-20 mg/ml and crystallized. The yield of FKBP Ile90PCL was 120 mg/L (crude). Crystallized FKBP Ile90PCL was used to obtain the X-ray structure of the PCL-containing protein. 19A-C show SDS-PAGE and mass spectrometry data and crystallization results for PCL biosynthetically incorporated in FKBP12. The mass obtained was 12085.6 Da, which is consistent with 12084 Da, the expected value for single site incorporation of PCL.

The sequence of FKBP12 (SEQ ID NO: 24) with the PCL incorporation site (bold and underlined) is shown below:

Example 10 : Site-specific incorporation of biosynthetically produced PCL into fibroblast growth factor 21 (FGF21) using Escherichia coli cells.

In this example, the incorporation of PCL into 20 distinct TAG-encoded sites of FGF21, a human fibroblast growth factor, expressed from a second plasmid co-transformed with the same Escherichia coli cells is provided.

Fibroblast growth factor 21, FGF21 were expressed from a pSpeedET vector having an N-terminal tag (MGDSKIHHHHHHENLYFQG) (SEQ ID NO: 21) and coding residues 33-209 of the translated human protein (Klock, HE, Koesema EJ, Knuth MW, Lesley, SA, "Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts", Proteins. 2008 May 1;71(2):982-94). The TAG codon for PYL analog incorporation and subsequent PEG attachment was introduced at the following 20 separate positions of the FGF21 (SEQ ID NO: 25) aa33-209 construct: Ser35, Gln39, Arg47, Gln56, Arg64, Asp74, Lys84, Lys87, Lys97 , Arg100, Arg105, His115, Arg124, Glu129, Lys150, Arg154, Leu167, Leu170, Leu181 and Gln184. The sequence of the FGF21 (33-209 aa) construct is shown below along with the sites of incorporation (highlighted in bold and underlined) in 20 separate constructs.

HK100 or BL21(DE3) cells were co-transformed with pSpeedet-FGF21 plasmid and pAra-pylSTBCD and selected on LB+Kan+Cm. Liquid cultures were grown as in Example 8 and 5 mM D-ornithine was added prior to induction. _{The OD 595} for conversion to 30° C. was tested from 0.2 to 1.0, followed by induction with 0.2% arabinose or 1 mM IPTG at OD595 = 0.4-2.0 after 15-30 minutes. After induction, the cells were grown for approximately 20 hours and then harvested. Cells were lysed with 5% glycerol, 1% Triton X-114, or 2.5% deoxycholate in TBS. The insoluble pellet was then resuspended in TBS+6M guanidine-HCl (pH 8). Proteins were purified on Ni-NTA resin and refolded on or after eluting from the column. The tag was subsequently removed with TEV protease and the product was purified by Ni-NTA, ion exchange, and size exclusion chromatography.

Representative data showing the incorporation of PCL into FGF21 is shown in FIG. 20. Preliminary expression yields are reported in Table 4. Both full-length and truncated FGF21 proteins were purified as the construct was characterized as containing an N-terminal His-tag. The range in which the TAG codon is used as a stop codon (which produces a truncation protein) and the range used to incorporate PCL (which produces a full-length protein) was dependent on the different incorporation sites (FIG. 20 ). The expected yield of the desired full-length protein was 4 to 56 mg/L, but no FGF21 protein could be detected in three of the mutants (Table 4), and the total yield of the protein obtained from the remaining 17 mutants was 5.7. To 143 mg/L. PCL incorporation was confirmed by mass spectrometry for all mutants.

Example 11: mTNF -α by PCL mixing .

To express the mTNF-α Gln21PCL mutant, E. E. coli BL21(DE3) cells were co-transformed with the pAra-pylSTBCD and individual mutant mTNF-α genes on the pET22b plasmid vector. Transformed cells were grown at 37° C. in TB medium in the presence of 5 mM D-ornithine, and when OD ₆₀₀ reached 0.5, they were induced with 1 mM IPTG and 0.2% (w/v) arabinose. Subsequently, the cells were continuously shaken at 30° C. for 12-16 hours and harvested. Cell pellets were stored at -20°C until use. The X-ray crystal structure of mTNF-α is the N-terminal His ₆ tag followed by the PCL incorporation sites Lys11 and Gln21 as shown below in the protein sequence (SEQ ID NO: 26) of the recombinant mTNF-α containing the factor Xa cleavage site (GIEGR). Indicated:

Example 12 : Incorporation of PCL into mEGF.

To express the mEGF-Tyr10PCL mutant, E. E. coli BL21(DE3) cells were co-transformed with the pAra-pylSTBCD and individual mutant mEGF genes on the pET22b plasmid vector. Transformed cells were grown at 37° C. in TB medium in the presence of 5 mM D-ornithine, and when OD ₆₀₀ reached 0.5, they were induced with 1 mM IPTG and 0.2% (w/v) arabinose. Subsequently, the cells were continuously shaken at 30° C. for 12-16 hours and harvested. Cell pellets were stored at -20°C until use. The X-ray crystal structure of mEGF showed PCL incorporation sites Tyr10 and Tyr29 as shown below in the protein sequence (SEQ ID NO: 27) of the recombinant mEGF with a _{C-terminal His 6 tag:}

Example 13: mTNF -α to the new pyrrolidine (Pyl), or incorporation of the PCL.

To insert PYL into mTNF-α, E. E. coli BL21(DE3) cells were converted to the mutant mTNF-α gene on the pET22b plasmid vector in which the codon for Gln21 (CAA) was mutated to the stop codon (TAG), as well as M. mazei pylS, pylT, pylB, It was co-transformed with pAra-pylSTBCD containing pylC, and pylD. To incorporate only PCL into the mutant mTNF-α, pAra-pylSTBCD was replaced with pAra-pylSTCD lacking the gene for the putative methyl transferase PylB. Transformed cells were grown at 37° C. in TB medium in the presence of 5 mM D-ornithine, and when OD ₆₀₀ reached 0.5, they were induced with 1 mM IPTG and 0.2% (w/v) arabinose. Subsequently, the cells were continuously shaken at 30° C. for 12-16 hours and harvested. The cell pellet was stored at -20°C. After thawing the cell pellet on ice for 15 minutes, the cells were resuspended in lysis buffer (20 mM Tris/HCl, 50 mM NaCl, pH 8.0) at 3 ml per gram wet weight. Lysozyme was added at 1 mg/ml, and the cells were sonicated for 2 minutes on ice. The lysate was centrifuged at 30,000 xg for 20 minutes at 4° C. to pellet the cell debris. 1 ml of a 50% Ni-NTA slurry (Qiagen) was added to the cleared lysate and mixed gently by shaking at 4° C. for 60 minutes. The lysate-Ni-NTA mixture was loaded onto the column and the flow-through was collected. After washing the resin with 20 mL of 25 mM imidazole in PBS (pH 8.0), the protein was eluted with 2.5 ml of 250 mM imidazole in PBS (pH 8.0), and by using a PD-10 column (GE Healthcare). The buffer solution was exchanged with PBS, pH 7.4. Expression of mTNF-α Gln21TAG with or without pylB, as well as with or without D-ornithine was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (FIG. 21A ). In Figure 21A, lane 1 is the molecular weight of the standard SeeBlue Plus2 prestained standard; Lane 2 is expression in the presence of both pylB and D-ornithine; Lane 3 is expression in the presence of pylB without D-ornithine added; Lane 4 is expression in the absence of pylB with 5 mM D-ornithine added; Lane 5 is expression in the absence of both pylB and D-ornithine. The data show that the expression of full length mTNF-α is dependent on the presence of D-ornithine and is similar in the absence or presence of the pylB gene.

Example 14 : Incorporation of pyrrolysine (Pyl) or PCL into mEGF

this. E. coli BL21(DE3) cells were co-transformed with the individual mutant mEGF Tyr10TAG and Tyr29TAG genes on the pAra-pylSTBCD and pET22b plasmid vectors. Both constructs were expressed at 37° C. in TB medium in the presence of 5 mM D-ornithine, and 1 mM IPTG and 0.2% (w/v) arabinose were added when _{OD 600 reached 0.5.} Then, the temperature was lowered to 30° C., and cells were harvested 16 hours after induction. The cell pellet was resuspended in 20 mL of 20 mM Tris/HCl (pH 8.5) and sonicated for 5 minutes. After centrifugation at 30,000 xg for 20 minutes, the supernatant was decanted. The pellet was resuspended in 20 mL of 20 mM Tris/HCl (pH 8.5) containing 2% (v/v) Triton-X100 by sonication. After another round of centrifugation at 30,000 xg for 20 minutes, the pellet was dissolved in 10 mL of 8 M urea, 20 mM Tris/HCl, 10 mM β-mercaptoethanol, pH 8.5 by sonication. Insoluble cell debris was removed by centrifugation (30,000 xg, 20 min), and the supernatant was refolded in buffer (100 mM Tris/HCl, 4 mM reduced glutathione, 0.4 mM glutathione oxide, 20% (v/v) ethanol, pH. 8.5) was diluted 2 times. The diluted samples were then dialyzed against refolding buffer at 4° C. overnight using a slide-a-riser dialysis cassette (3500 Da molecular weight cutoff, Pierce). Insoluble protein was removed by centrifugation at 30,000 xg for 20 minutes, and the supernatant was supplemented with β-mercaptoethanol to a final concentration of 2 mM. 1 ml of 50% Ni-NTA slurry (Qiagen) was added to the refolded protein and mixed gently by shaking at 4° C. for 60 minutes. The protein-Ni-NTA mixture was loaded onto the column and the flow through was collected. After washing the resin with 20 mL of 25 mM imidazole in PBS (pH 8.0), the protein was eluted with 2.5 mL of 250 mM imidazole in PBS (pH 8.0). Finally, the protein was buffer exchanged with PBS (pH 7.4) by using a PD-10 column (GE Healthcare). The purity of the protein sample was investigated by SDS-PAGE (Fig. 21B). In FIG. 21B, lane 1 is the molecular weight of the standard Cblue Plus2 pre-stained standard, and lane 2 is the mEGF Tyr10TAG mutant protein after Ni-NTA purification.

In addition, as obtained using the method for mTNFα, mEGF Tyr10TAG, ESI-MS spectra were obtained for the protein obtained in the presence or absence of PylB expression (FIG. 21C). In Figure 21C, the lower mass spectrum shows that PYL incorporation occurs predominantly in the presence of the pylB gene (expected mass of mEGF Tyr10Pyl = 7310 Da), and the upper mass spectrum in Figure 21C shows that the only PCL incorporation occurs in the absence of pylB. (Expected mass of mEGF Tyr10Pyl = 7296 Da). As such, of the proteins observed in FIG. 21B, lane 2 was mEGF mixed with PYL (confirmed by additional LC-MS/MS analysis). Similarly, of the proteins observed in FIG. 21A, lane 2 will probably be mTNFα with PYL incorporated, and lane 4 will probably be mTNFα with PCL incorporated.

Additionally, LC-MS/MS extracted ion chromatogram PCL/PYL from mass spectrometry of incorporation of PCL and PYL into mEGF Tyr10TAG samples expressing all genes (M. majay pylS, pylT, pylB, pylC, and pylD). Quantification of the ratio showed that PYL was 5 to 10 times more abundant than PCL, while LC- of PCL and PYL incorporation into mTNF Gln21TAG samples expressing all genes (M. majei pylS, pylT, pylB, pylC, and pylD). Quantification of the PCL/PYL ratio from MS/MS extracted ion chromatogram mass spectrometry showed that PCL was about 7 times more abundant than Pyl. Quantification in the absence of the PylB gene revealed only PCL protein, indicating that PYL incorporation was absolutely dependent on pylB, further suggesting that PylB is actually a methyltransferase required for the biosynthesis of Pyl.

Example 15 : Incorporation of other analogs and precursors :

HK100 cells (derived from Genehogs; Invitrogen) were co-transformed with pAra-pylSTBCD, pAra-pylSTCD, pAra-pylSTC, pAra-pylSTD, or pSUPAR-pylST and pMH4-FASTE-L2222TAG-L2223I. . Cells were grown in 25 ml cultures of Terrific Broth (TB) (Sigma) at 37° C. to an OD595 of about 0.6. Cells were transferred to 30° C. and analog or precursor compounds were added to each individual culture at the concentrations shown in FIG. 15. The evaluated compounds were N-ε-cyclopentyloxycarbonyl-L-lysine (CYC; Sigma), D-ornithine (Chem-Impex), PCL-A (see Example 36-1, Compound 3647-125). , PCL-B (see Example 36-2, compound 3793-011), P2C (see Example 36-2, compound 3647-164), P5C (see Example 35-1, compound 3793-007) and Lys- It was Nε-D-ornithine (see Example 35-2, compound 3793-031). The cells were then induced with 0.2% arabinose after 20 minutes. After 18-20 hours, cells were harvested by centrifugation. Cells were lysed by sonication, purified on Ni-NTA (Qiagen) under natural conditions and evaluated by SDS-PAGE gel followed by Coomassie staining (0.25 ml Ni-NTA, 0.75 ml elution, 20 μl on gel) I did. 15A shows a protein (FAS-TE) grown in the presence of the indicated pyl gene (pylS/pylT or pylB/pylC/pylD/pylS/pylT) and purified from cells fed the indicated compound. Cyc and D-ornithine (D-Orn) were used as positive controls. No compound was added as a negative control. The gel sample volumes in lanes 1-11 and 12-18 were internally consistent, respectively.

Lanes 2 and 3 only show the pylS and pylT genes and proteins obtained from cells grown with PCL-B (Lys-P2C) or PCL-A (Lys-P5C), respectively. Both compounds were incorporated into the protein, showing that PylS can use both compounds. Lanes 5 to 11 show the precursor Lys-Nε-D-Orn (lane 10) in cells containing the entire set of pyl biosynthesis genes (pylB/pylC/pylD/pylS/pylT), or a precursor with only a pyrroline ring: P2C ( It shows the evaluation of protein biosynthesis in the presence of lane 6) and P5C (lane 8). Precursors with only pyrroline rings: P2C (lane 6) and P5C (lane 8) were not sufficient to support PCL biosynthesis, suggesting that they are not intermediates of the pathway. However, Lys-Nε-D-Orn (lane 10) expressed a protein in which PCL was incorporated into the amber codon, indicating that it is an intermediate of the PCL biosynthetic pathway. Table 5 shows the amount of protein obtained (Bradford), the observed mass and the ratio of PCL:PYL:CYC obtained.

To determine whether Lys-Nε-D-Orn is an intermediate upstream of the biosynthetic pathway of any of the required genes (pylC and pylD), HK100 cells (derived from Genehox; Invitrogen) were converted to pSUPAR-pylST, pMH4. Cotransformed with -FASTE-L2222TAG-L2223I, and pAra-pylSTB, pAra-pylSTC, pAra-pylSTD, pAra-pylSTCD or pAra-pylSTBCD. Figure 15B showed that only pylD (lane 15) is required to form PCL from Lys-Ne-D-Orn, suggesting that it is an upstream intermediate of pylC in the biosynthetic pathway. In addition, it was confirmed that Lys-Ne-D-Orn is a substrate of PylD by NMR evaluation.

Example 16 : Derivatization of PCL incorporated in hRBP4 with 2-amino benzaldehyde , 2-amino- acetophenone and 2-amino-5-nitro-benzophenone.

This example provides for labeling PCL with 2-amino-benzaldehyde (2-ABA), 2-amino-acetophenone and 2-amino-5-nitro-benzophenone. The reaction appears to follow the general scheme shown in Figure 22. The structure formed was evaluated using mass spectrometry and NMR. 23 shows protein conjugates formed from three different 2-amino-benzaldehyde residues and provides the expected and observed mass changes.

hRBP4 2- in ABA Site-specific modified by PCL Mass spectrometry detection of

Retinol binding protein (hRBP4) was expressed in HEK293F cells as described in Example 2. PCL was incorporated instead of Phe122 (hRBP4 mutant #6), which was confirmed by mass spectrometry (FIGS. 6A-11). 10 μl of hRBP4 Phe122PCL stock solution was mixed with 89 μl of 200 mM sodium acetate buffer (pH 5.0) and 1 μl of 1 M 2-ABA solution and incubated at room temperature for 16 hours. The final concentration in the reaction mixture was 17 μM hRBP4 Phe122PCL protein and 10 mM 2-amino-benzaldehyde (2-ABA). The mass spectrum of the derivatized hRBP4 is shown in FIG. 24, and the mass obtained at this time corresponds to the 2-ABA adduct of PCL (23269.2 Da). Unmodified hRBP4 had a mass of 23166.8 Da, so the observed 102.4 Da mass increase (expected + 103 Da) indicates that hRBP4 was modified by 2-ABA. The peak intensity of at least 96% in the mass spectrum is due to the 2-ABA adduct of PCL, indicating that the reaction is almost complete.

LC-MS analysis of trypsin digestion of 2-ABA-derivated hRBP4 Phe122PCL protein was performed, and YWGVASF ^* LQK peptide (SEQ ID NO: 17) ^{expected by MS/MS analysis (Fig. 25A) was confirmed (where F *} is Fig. It has a mass that matches the mass of the 2-ABA-modified PCL as shown in 23). The reaction was complete with no detection of non-derivatized PCL residues. The assigned MS/MS spectrum of YWGVASF ^* LQK (F ^* = PCL-2-ABA adduct) is provided below:

Figure 25B is ^{the TIC and EIC of the 2+ ions of YWGVASF *} LQK (F ^* = PCL and PCL-2-ABA adduct), EIC (extracted ion chromatogram) derivatized and derivatized (not detected). ) Comparison with the species indicated that the reaction was complete. Figure 25C is a mass spectrometric analysis of hRBP4 Phe122PCL derivatized by 2-ABA, ^{where the 3+ and 2+ precursors of YWGVASF *} LQK were shown at m/z 459.92 (3+) and 689.37 (2+), respectively. (F ^* = PCL-2-ABA adduct). This example demonstrated that the observed reaction by 2-ABA occurred site-specifically, and that the PCL residue was incorporated into the desired TAG site of residue 122.

pH Reaction efficiency as a function of:

The reaction efficiency as a function of pH, (FIG. 26 ), shows that 17 μM hRBP4 PCL mutant protein was reacted with or without 10 mM 2-amino-benzaldehyde (2-ABA) in 200 mM sodium acetate buffer (pH 5.0), Alternatively, it was evaluated by reacting with 10 mM 2-amino-benzaldehyde (2-ABA) 10x PBS buffer (pH 7.4) for 12 hours at room temperature. The mass spectrum of the reaction mixture indicated that a total peak intensity of at least 87% corresponds to the peak intensity of the protein modified by one 2-ABA moiety (+102 Da as expected) (Figures 26A, B and C). . The pH 7.4 reaction mixture contained approximately 13% unreacted protein (Figure 26C), whereas only 4.2% unreacted protein was detected in the reaction at pH 5.0 (Figure 26B), which at pH 5.0 compared to pH 7.4. This suggests that the reactivity of PCL is slightly higher. Since hRBP4 with OMePhe incorporated instead of Phe62 was not modified by the presence of 10 mM 2-ABA, the reaction was specific for the presence of PCL (FIGS. 26D, E and F).

The reaction efficiency as a function of the protein concentration of the reactants and the reaction efficiency with other 2- ABA -like residues :

17 μM hRBP4 PCL mutant protein was added to 0.1 mM 2-amino-benzaldehyde (2-ABA), 0.1 mM 2-amino-acetophenone (2-AAP) or 0.1 mM 2-amino in 200 mM sodium acetate buffer (pH 5.0). By reacting with -5-nitro-benzophenone (2-ANBP), the reaction efficiency as a function of the protein concentration ratio of the reactants and the reactivity with 2-ABA-like reactants were evaluated. After 12 hours at room temperature, the mass spectrum of the reaction mixture showed the expected mass of the conjugated protein (FIG. 27 ). For 2-ABA, the relative intensity of the correctly bonded peak was 88% of the total intensity; Only 4.2% of the protein remained unreacted (FIG. 27A ). For 2-AAP, 93% appeared to be reacted; 4.5% did not react (FIG. 27B). In the case of 2-ANBP, only 5.4% reacted (FIG. 27C ), which appears to be due to the low solubility of the reactive reagent, i.e. 2-ANBP precipitates immediately upon addition to the solution containing the hRBP4 PCL mutant protein.

This example shows that the PCL modified protein reacts with other 2-amino-benzaldehyde analogues and is derivatized at the site where PCL is incorporated. In all cases, the measured mass of the modified protein coincided with the mass expected in the structure shown in FIG. 23. The data also showed that the conjugation reaction proceeded with high efficiencies and was nearly complete at only a low reactant to protein ratio of 6 to 1. The data also showed that each protein sample was derivatized only once. However, it was observed that at a very high reactant to protein ratio (4700 fold) and pH 7.5, multiple reactant molecules were attached (FIG. 28A ). Precipitation of the same sample at pH 5.0 suggests that there are also multiple reactions. Similarly, hRBP4 (6.5 μM) modified with OMePhe instead of Phe62 showed a conjugation pattern similar to that observed in FIG. 28A when reacted with a 15400-fold molar excess of 2-ABA at pH 7.5 (FIG. 28B ). This indicates that adhesion under these conditions (molar excess of reactants greater than protein) is not dependent on the presence of PCL side chains, but will probably involve lysine side chains. However, the example reactions show that the PCL moiety incorporated in the protein can be specifically and almost quantitatively derivatized by reaction with a 2-amino-benzaldehyde-containing molecule added in a small molar excess.

Example 17 : Derivatization of PCL incorporated in FAS - TE with 2-amino-acetophenone

As in Example 8, 16 μM of FAS-TE Tyr2454PCL produced in Escherichia coli was reacted with 1 mM 2-AAP for 16 hours at room temperature, and reacted in 200 mM sodium acetate buffer at 4° C. for 24 hours at pH 5.0. Made it. Figure 29A shows the mass spectrum of unreacted FAS-TE Tyr2454PCL, and Figure 29B shows the mass spectrum of the reaction mixture: 100% of the observable peak intensity is 116.8 Da greater than the mass of the unreacted material, 2-AAP modified It occurred at 33318.8 Da, which is within the error of the 116 Da mass increase expected in FAS-TE Tyr2454PCL. Similarly, at pH 7.4 the reaction was 95% complete (Fig. 29C (unreacted) and Fig. 29D (reacted)).

Example 18: in the PCL incorporated in hRBP4 2- amino-acetophenone-derivative by PEG8 Chemistry

This example shows that PCL incorporated in hRBP4 can be completely derivatized at a single site by a polyethylene glycol (PEG) derivative of 2-amino-acetophenone. In this example, PEG contains 8 ethylene glycol units, and its structure is shown in FIG. 31. This example also shows that at pH 5.0 and pH 7.5, wild-type hRBP4 does not react with 2-AAP-PEG8 with reagents of up to 2300 per protein ratio.

hRBP4 mutant #6 was prepared as in Example 2. 2-Amino-acetophenone PEG8 (TU3205-044) was prepared as described in Example 20. After allowing the reaction to proceed at room temperature for 14 hours and at 4° C. for 72 hours, a mass spectrum for the reaction mixture was obtained.

When performing the reaction at pH 7.5, 10 µl of hRBP4 Phe122PCL (0.22 mg/mL, in PBS, pH 7.5) was diluted with 10 µl of 10x PBS. 0.2 μl or 2 μl of a 100 mM stock solution of 2-AAP-PEG8 (in water) was added at a final concentration of 1 and 9.1 mM, respectively. Protein concentrations were 4.7 μM and 4.3 μM, respectively, and the reactants were 210 or 2100 molar excesses for the protein. FIG. 32A shows the mass spectrum of the reaction mixture at a 210 to 1 ratio indicating that the reaction was approximately 95% complete, and the observed mass increase of 556 Da was the same as expected (FIG. 31 ). 100% completion was obtained in the reaction at 2100 molar excess (data not shown).

Similarly, the reaction was carried out at pH 5.0. In this case, 10 μl of hRBP4 Phe122PCL (0.22 mg/mL, pH 7.5 in PBS) was diluted with 90 μl of 200 mM sodium acetate buffer (pH 5.0). 1 μl or 10 μl of a 100 mM stock solution of 2-AAP-PEG8 (in water) was added to a final concentration of 1 and 9.1 mM, respectively. Protein concentrations were 0.94 and 0.86 μM, respectively, and the reactants were 1050 or 10500 molar excesses for the protein. 32B shows the mass spectrum of the reaction mixture at 1050 to 1 ratio. 100% completion was obtained for both reactions and the observed mass increase of 556 Da was the same as expected (Figure 31).

To test the reactivity of 2-AAP-PEG8 with the wild-type hRBP4 protein, the test reaction was set up similar to the above. For the pH 7.5 reaction, the final wild-type protein concentration was 20 μM, and the 2-AAP-PEG8 concentration was 1 or 9.1 mM, resulting in a molar ratio of 46 and 460 to 1. The pH 5 reaction was performed at 4 μM protein concentration and 1 and 9.1 mM 2-AAP-PEG (230 and 2300 to 1 reactant to protein). In all four reactions, only unmodified wild-type hRBP4 protein was observed at the expected mass (Fig. 32D-F). This example shows that the coupling reaction of 2-AAP-PEG is very specific for the presence of PCL residues in the target protein.

Example 19 : Derivatization of PCL incorporated in FAS - TE with 2-amino-acetophenone-PEG of various molecular weights

This example shows the universality of the reactivity of the PCL side chain with 2-amino-acetophenone PEG. This example further shows that 2-AAP-PEG of sufficient length, useful for modifying biotherapeutic proteins, can be conjugated to PCL modified proteins.

As described in Example 8, 16 μM of FAS-TE Tyr2454PCL produced in Escherichia coli was prepared in 1 mM TU3205-044 (2-AAP-PEG8) and 200 mM sodium acetate buffer for 16 hours at pH 5.0 at room temperature. Reacted. Figure 33A shows the mass spectrum of unreacted FAS-TE Tyr2454PCL, and Figure 33B shows the mass spectrum of the reaction mixture: In Figure 33B, 100% of the observable peak intensity is at 33758.4 Da, which is 556 Da greater than the mass of the unreacted material. Occurred. This mass difference is as expected for the 2-AAP-PEG8 modified FAS-TE Tyr2454PCL (Fig. 31).

In a further example, PCL incorporated into Tyr2454 of FAS-TE was derivatized with three different 2-AAP-PEGs with MW of 0.5 kDa, 2.4 kDa and 23 kDa. FAS-TE single site PCL mutants were generated in Escherichia coli in a yield of approximately 160 mg/L (corresponding to a yield of approximately 80% by weight) as described in Example 8. Aliquots of FAS-TE Tyr2454PCL (0.16 mM in PBS, pH 7.5) were added to TU3205-044 (0.5 kDa 2-AAP-PEG), TU3205-048 (2.4 kDa 2-AAP-PEG) and TU3205-052 (23 kDa 2 -AAP-PEG) and 10:1 or 100:1 were reacted for 6 days at 4°C or room temperature. The structure of PEG and its synthesis are described in Example 37.

Prior to SDS-PAGE analysis (FIG. 34 ), His-tagged FAS-TE protein was bound to Ni-NTA beads to remove excess PEG reagent, and the beads were washed repeatedly with PBS buffer. For gel and mass spectrometric analysis, excess buffer was removed from the Ni-NTA beads and the protein was eluted with imidazole buffer. Specifically, 50 μl of Ni-NTA beads were added to 50 μl of the reaction mixture, incubated for 2 hours, and the reaction mixture and buffer were separated by centrifugation. Then, the beads were washed 3 times with 1 mL PBS; 70 μl of 250 mM imidazole buffer, 20 mM Tris, pH 8 were added to the beads to elute the gel and protein for mass spectrometry analysis. PEGylation with 0.5 kDa 2-AAP-PEG could not be analyzed by SDS-PAGE, but was confirmed by mass spectrometry. The degree of reaction was approximately 57% for a 100:1 room temperature reaction and 43% for a 100:1 4°C reaction (data not shown). In the case of the larger 2-AAP-PEG, the PEGylated product could be analyzed by SDS-PAGE (FIG. 34 ). All reactions were incomplete and proceeded approximately 25-30% for 2.4 kDa and 23 kDa 2-AAP-PEG. The degree of single site conjugation and reaction at the PCL site was confirmed by mass spectrometry in the case of 2.4 kDa 2-AAP-PEG (FIG. 33). Mass spectrometric data for the 23 kDa 2-AAP-PEG derivatized protein could not be obtained because the PEG was not homogeneous.

The reported reaction yield is a lower estimate because the stringency of the Ni-NTA bead extraction can aid in the extraction of unreacted FAS-TE, and PEGylation can lower the affinity of the His-tag (PEG of FAS-TE Leu2222PCL). In case of anger; data not shown). However, detection of the PEGylated material after extraction showed that the PCL-2-AAP-PEG linkage was stable.

Example 20 : Derivatization of PCL incorporated in FGF21 with 2-amino-acetophenone- PEG of different molecular weight

This example shows that FGF21, in which PCL is incorporated at various positions, can be PEGylated by 2-AAP-PEG reagent. This example also shows that PEGylated FGF21 mutants can be separated from unreacted full-length FGF21 and truncated FGF21 by a combination of ion exchange and size exclusion chromatography.

FGF21 with PCL incorporated at position 84 of lysine was expressed in Escherichia coli, refolded, and purified as described in Example 10, except that the protein was not cleaved with TEV protease. The protein stock was approximately 6.6 mg/mL in PBS and contained approximately 20% FGF21 protein truncated at residue 84. 5 μl of FGF21 stock solution was mixed with 45 μl 200 mM sodium acetate buffer (pH 5.0) and 0.5 μl of 100 mM TU3205-044 (2-AAP-PEG8, see Example 20) stock solution. The final molar ratio was 1 mM 2-AAP-PEG8 to approximately 30 μM FGF21. The reaction proceeded for 16 hours at room temperature and allowed to complete.

Figure 35A shows the mass spectrum of unreacted FGF21, and Figure 35B shows the mass spectrum of the reaction mixture: the PEGylation reaction proceeded to completion and produced a protein of 21792.4 Da, which was 556 Da larger than the mass of the unreacted material. This mass difference is expected for the 2-AAP-PEG8 modified FGF21.

Various FGF21 PCL mutants (expressed and purified as in Example 10) were PEGylated with 23 kDa 2-AAP-PEG (see TU3205-052, Example 37-3). Typically the protein concentration was between 100 and 400 μM, and 23 kDa 2-AAP-PEG was added in PBS buffer (pH 7.4) to a final concentration of 1 mM. The reaction was incubated at 4° C. for 3 days. SDS-PAGE of the PEGylation reaction with 7 representative FGF21 PCL mutants is shown in Fig.36A, and 8 types of purified PEGylated FGF21 proteins isolated from full-length (FL) unreacted FGF21 and truncated (TR) FGF21. Is shown in Figure 36B.

Example 21: in the PCL incorporated into the EPO 2- amino-acetophenone-derivative by PEG Chemistry

PCL was incorporated into various locations of mouse EPO as described in Example 6. After Ni-NTA purification, mEPO constructs (#6, #9 and #10 mutant constructs) were further purified in PBS by S-300 gel filtration column. The purified mEPO protein was concentrated to approximately 1 mg/ml. Activated 23 kDa 2-AAP-PEG (TU3205-052, see Example 37-3) was added to the purified protein at 1 mM, and incubated under the conditions shown in Table 6.

All mEPO constructs were run as monomers on the column. When 23 kDa 2-AAP-PEG was incubated with the purified protein, mEPO was PEGylated at a single site because it moved to the 65 kDa band of SDS-PAGE (FIG. 37). The efficiency of the reaction varied from 10% to 15% depending on the conditions, and was PEGylated to a higher degree at pH 5.5 than at pH 7 and pH 8.5. In addition, temperatures higher than 4° C. did not significantly increase the degree of PEGylation.

Derivatization of the PCL only D- mixed in TE by industrial Min - hRBP and FAS: Example 22.

This example shows that the amino sugar, D-mannosamine, is directly coupled to PCL incorporated into two target proteins. This example presents a general reaction scheme for the glycosylation of PCL-containing proteins (Figure 38).

Human RBP4 Phe122PCL (mutant #6) was prepared in HEK293F cells as in Example 2. 10 μl of 170 μM protein stock solution was added to 89 μl 10×PBS buffer (pH 7.5) and mixed with 1 μl of 1 M D-mannosamine. hRBP4 Phe122PCL protein (17 μM) was incubated with 10 mM D-mannosamine at room temperature for 14 (no reaction was observed), followed by incubation at 37° C. for 48 hours. 39 shows the mass spectrum of the reaction mixture after the 37° C. incubation period. In addition to the expected peak of unreacted hRBP4 at 23165.6 Da (estimated 23166 Da), a presumed assigned peak for the D-mannosamine adduct was observed at 23300.0 Da. The mass increase of 164.4 Da, although close, was not equal to the expected increase of 161.1 Da for the putative reaction product shown in Figure 38. In addition, a part of the species-degraded protein was detected at 21007.8 Da.

In the second example, FAS-TE (11 μM) modified by PCL at position 2222 expressed in Escherichia coli and purified as in Example 8 was mixed with 10 mM D-mannosamine at room temperature for 72 hours. Incubated at. The mass spectrum of the unreacted sample is shown in Figure 40A, which contained the expected signal at 33250.4 Da corresponding to the unreacted protein, and the peak at 33360.4 Da was due to protein contamination in the sample. Mass spectrometry of the reaction mixture showed the expected signal at 33250.4 Da corresponding to the unreacted protein (FIG. 40B ). An additional peak was found at 33408.8 Da, which is 158.4 Da larger than the starting material, which is presumed to be the peak of the reaction product shown in Figure 37. Comparison of the two samples suggests that FAS-TE Leu2222PCL was converted to a D-mannosamine adduct in a yield of approximately 50% under the above reaction conditions.

Example 23: PCL-mediated cross-linking of FGF21 shares

This example shows that proteins can be covalently dimerized through a bifunctional PCL-specific cross linker. FGF21 with PCL incorporated at position 84 of lysine was expressed in Escherichia coli, refolded, and purified as described in Example 10, except that the protein was not cleaved with TEV protease. FGF21 Lys84PCL was derivatized with cross linker TU633-010 (Figure 43A; see Example 37-4 for synthesis). The protein stock was approximately 6.6 mg/mL in PBS and contained approximately 20% FGF21 protein cleaved at residue 84. 5 μl of FGF21 stock solution was mixed with 45 μl 200 mM sodium acetate buffer (pH 5.0). 0.1 μl of a 5 mM stock solution of cross linker TU633-010 (in DMSO) was added to a final concentration of 10 μM cross linker and approximately 30 μM FGF21. Similarly, a stock solution of FGF21 (pH 7.4) in 50 μl of PBS was reacted with 1 μl of 5 mM TU633-010 at a concentration of 100 μM of cross linker and approximately 300 μM of FGF21. A sample of FGF21 diluted with 200 mM sodium acetate buffer was prepared as a control and treated in the same manner. After 16 hours at room temperature, aliquots of the reaction and control samples were analyzed by mass spectrometry and SDS-PAGE analysis without purification. The mass spectrum obtained for the pH 5.0 sample (Fig. 43B) shows the expected mass of the covalent dimer (43037.2 Da; relative intensity of all FGF21 peaks 53%), the expected mass of FGF21 with a cross linker attached at one end (21820.0 Da; 33). %) and unreacted FGF21 (21235.2 Da; 14%). For the pH 7.4 sample, no covalent dimers were detected; 28% of FGF21 reacted single with the cross linker, whereas most proteins did not (data not shown).

Adjustment of the reactants to protein concentration further increased the yield of covalent dimers. Specifically, 10 μl of FGF21 stock solution was mixed with 90 μl of 200 mM sodium acetate buffer (pH 5.0). A 5 mM stock solution of 0.3 μl of cross linker TU633-010 (in DMSO) was added to a final concentration of 15 μM cross linker and approximately 30 μM FGF21. One sample was incubated at room temperature for 4 days, and the second sample was incubated at 4°C. Further, a sample in 10x PBS (pH 7.5) was prepared and incubated in the same manner. For the pH 5.0 sample incubated at room temperature, the peak of the covalent FGF21 dimer at the expected mass of 43034.4 Da was the dominant species, and unreacted FGF21 was not detected at 21233.6 Da. Some FGF21 modified by attaching a cross linker to one end was detected at 21818.4 Da (FIG. 44A). The reaction did not progress to the same extent as at pH 5.0 and 4° C., leaving approximately 19% of FGF21 unreacted; 40% was modified by attaching a cross linker at one end, while the mass spectrum peak intensity of approximately 41% was that of the covalent dimer. The reaction at pH 7.5 did not produce any covalent dimers as indicated by SDS-PAGE (FIG. 44B ).

Site-specific modification of pyrroline - carboxy -lysine ( PCL ) containing proteins by various other molecules.

In another embodiment, the coupling of a 2-aminobenzaldehyde (ABA) conjugate provided herein and a 2-aminoacetophenone (AAP) conjugate to a PCL-containing protein is in 10 x phosphate buffered saline (PBS) (pH 7.0). It was carried out at 25 degreeC. The conjugation reaction was initiated by the addition of 10 μM PCL-containing protein and 100 μM ABA/AAP conjugate. The complete formation of the protein conjugate was confirmed by electrospray ionization-mass spectrometry (ESI-MS) or matrix assisted laser desorption/ionization (MALDI). Coupling of ABA/AAP DNA conjugates was analyzed by gel transfer assay using a NuPAGE 4-12% Bis-Tris gel (Invitrogen, Carlsbad, CA). After quantitative coupling, the protein conjugate is dialyzed with 10 mM sodium phosphate buffer (pH 7.5) and an Amicon Ultra-4 centrifugal filter unit with a cutoff of 10 kDa (Millipore Corporation) , And concentrated to 100 μM using Bedford, MA). Subsequently, a newly prepared 200 mM NaCNBH ₃ solution (dissolved in 10 mM phosphate buffer, pH 7.5) was added to a final concentration of 20 mM. After allowing the reduction reaction to proceed at 25° C. for 2 to 4 hours, 6 volumes of 10 mM sodium phosphate buffer (pH 7.5) were added to quench the reaction. Using a NAP-5 column or a PD10 column (GE Healthcare, Piscataway, NJ), the reduced protein conjugate was finally buffer exchanged with the desired buffer. Examples of non-limiting coupling of such 2-aminobenzaldehyde (ABA) conjugates and 2-aminoacetophenone (AAP) conjugates to various proteins are provided below.

Example 24 : Coupling of biotin reagent

In order to confirm whether biotin can be coupled to a protein in which one or more PCL residues are incorporated therein, mEGF-Tyr10PCL (see Example 12) was biotinylated using an ABA-biotin reagent (X3626-140, Example 40). And conjugated. Coupling of biotin is as follows, 500 μM ABA-biotin is added to 10 μM mEGF-Tyr10PCL in phosphate buffered saline (PBS, pH 7.0) and 0.5% (v/v) DMSO and reacted at 25° C. for 16 hours. Was carried out. Complete formation of the biotin conjugate was confirmed by ESI-MS (Figure 47A; expected mass for unreacted protein = 7296; expected mass for coupled protein = 7902).

After quantitative coupling, a freshly prepared 200 mM NaCNBH ₃ solution (dissolved in PBS, pH 7.0) was added to a final concentration of 20 mM. After allowing the reduction reaction to proceed at 25° C. for 3 hours, 6 volumes of PBS (pH 7.0) were added to quench the reaction. Excess NaCNBH ₃ was removed by dialysis against PBS (pH 7.0) at 4° C. using a Slide-A-Riser dialysis cassette (3,500 Da molecular weight cutoff, Pierce). The reduced biotin conjugate was concentrated using an Amicon Ultra-4 centrifugal filter unit (Millipore Corporation) with a cutoff of 3.5 kDa. After electrophoresis through NuPAGE 4-12% Bis-Tris gel (Invitrogen), biotinylated protein was transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot gel transfer system (Invitrogen). Moved to. Subsequently, the biotin conjugate was detected with horseradish peroxidase (HRP) conjugated to goat anti-biotin antibody (1:100 dilution, Cell Signaling Technologies), and Hyperfilm ECL (GE Health Care) was used (Fig. 47B). Uncoupled mEGF-Tyr10PCL and fluorescein-conjugated mEGF-Tyr10PCL served as negative controls. Lane 1, 20 pmol mEGF-Tyr10PCL-ABA-biotin conjugate; Lane 2, 8 pmol mEGF-Tyr10PCL-ABA-biotin conjugate; Lane 3, 2 nmol mEGF-Y10PCL; Lane 4, 20 pmol mEGF-Tyr10PCL-ABA-fluorescein conjugate.

Example 25 : Fluorescent molecule coupling

In order to confirm whether a fluorescent molecule can be coupled to a protein in which one or more PCL residues are incorporated therein, mEGF-Tyr10PCL (Example 12) using an ABA-fluorescein reagent (3793-050, see Example 42). Reference) was conjugated with fluorescein. Coupling of fluorescein is as follows, 1 mM of ABA-fluorescein is added to 10 μM mEGF-Tyr10PCL in phosphate buffered saline (PBS, pH 7.0) and 0.5% (v/v) DMSO, and at 25° C. It was carried out by reacting at for 16 hours. The formation of the fluorescein conjugate was confirmed by ESI-MS (Figure 47C; expected mass for uncoupled protein = 7296; expected mass for coupled protein = 8062).

The fluorescein conjugate _{was reduced with 20 mM NaCNBH 3} for 3 hours at 25°C. After quenching the reduction reaction with 6 volumes of PBS (pH 7.0), the remaining NaCNBH ₃ was removed by dialysis against PBS (pH 7.0) at 4°C using a slide-A-riser dialysis cassette (3,500 Da molecular weight cutoff). . The conjugate was then concentrated to 1 μM using an Amicon Ultra-4 centrifugal filter unit with a cutoff of 3.5 kDa. Absorption spectra in the 350-700 nm range of 1 μM mEGF-Tyr10PCL-ABA-fluorescein and 10 μM ABA-fluorescein were obtained using SpectraMax Plus (Molecular Devices). Obtained. They both showed maximum absorption at 500 nm.

Subsequently, the fluorescence spectrum was read on a Spectramax Gemini (GEMINI) fluorescence meter (Molecular Devices). Emission spectra for 1 μM mEGF-Tyr10PCL-ABA-fluorescein and 10 μM ABA-fluorescein, while keeping the excitation wavelength at 490 nm while scanning the emission wavelength from 510 nm to 750 nm with a step size of 2 nm. Obtained by. They both showed maximum emission at 522 nm. In addition, the excitation spectra for 1 μM mEGF-Tyr10PCL-ABA-fluorescein and 10 μM ABA-fluorescein keep the emission wavelength at 522 nm while the excitation wavelength is from 300 nm to 510 nm with a step size of 2 nm. It was obtained by scanning with.

Example 26 : Polysaccharide coupling

In order to confirm that the polysaccharide can be coupled to the protein in which one or more PCL residues are incorporated therein, mEGF-Tyr10PCL (implemented) using ABA-disaccharide (3793-050; Example 42, MW 546.52). Example 12) was conjugated with a disaccharide. The coupling reaction was performed by adding 1 mM ABA-disaccharide to 10 μM mEGF-Tyr10PCL mutant protein in PBS and 1% (v/v) DMSO (pH 7.0). The reaction was allowed to proceed at room temperature for 16 hours and analyzed by ESI-MS (Figure 47D). The mass spectrum showed a major peak at the expected mass (7825 Da) for the conjugated protein. The mass of the uncoupled protein was 7296 Da.

Example 27 : Immune modulator: mono- nitrophenyl Hapten conjugate coupling

In order to confirm whether the immunomodulatory agent can be coupled to the protein incorporated therein at least one PCL residue, mTNF-Gln21PCL and mTNF-Gln21PCL using ABA-mono-nitrophenyl hapten reagent (3793-001, Example 38-8) and mEGF-Tyr10PCL (see Examples 11 and 12) was conjugated with mono-nitrophenyl hapten. Coupling of the mono-nitrophenyl hapten conjugate was performed as described above. Figure 48A is the ESI mass spectrum of 3793-001 conjugated to mTNF-Gln21PCL (expected mass for uncoupled protein = 19275; expected mass for coupled protein = 19614), Figure 48B is mEGF-Tyr10PCL Is the ESI mass spectrum of 3793-001 conjugated to (expected mass for uncoupled protein = 7296; expected mass for coupled protein = 7635).

Example 28 : Immune modulator: di- nitrophenyl Hapten conjugate coupling

In order to confirm whether the immunomodulatory agent can be coupled to the protein incorporated therein at least one PCL residue, mTNF-Gln21PCL and mEGF-Tyr10PCL using di-nitrophenyl hapten (TU3627-088, Example 38-7). (See Examples 11 and 12) was conjugated with di-nitrophenyl hapten. Coupling of the di-nitrophenyl hapten conjugate was performed as described above. Figure 48C is the ESI mass spectrum of TU3627-088 conjugated to mTNF-Gln21PCL (expected mass for uncoupled protein = 19275; expected mass for coupled protein = 19688), Figure 48D is mEGF-Tyr10PCL Is the ESI mass spectrum of TU3627-088 conjugated to (expected mass for uncoupled protein = 7296; expected mass for coupled protein = 7709).

Example 29 : Immune Modulator: Coupling of TLR7 agonists

In order to confirm that the immunomodulatory agent can be coupled to a protein in which one or more PCL residues are incorporated therein, the ABATLR7 agonist reagent (X3678-114; see Example 38-3) was used to use mEGF-Y10PCL (Example 12). Reference) was conjugated with a TLR7 agonist. Coupling reactions were performed by adding 100 μM ABA-TLR7 agonist to 10 μM mEGF-Y10PCL mutant protein in 200 mM sodium acetate buffer and 1% (v/v) DMSO (pH 4.5). The reaction was allowed to proceed at room temperature for 16 hours and analyzed by ESI-MS (Figure 49A). The mass spectrum showed a major peak at the expected mass (8763 Da) for the conjugated protein, and the mass of the uncoupled protein was 7296 Da.

Example 30 : Immune Modulator: Coupling of PADRE Peptide

In order to confirm that the immunomodulator and peptide can be coupled to the protein with one or more PCL residues incorporated therein, mTNF-Gln21PCL and mEGF-Tyr10PCL (see Examples 11 and 12) were conjugated with the PADRE peptide. Coupling of the PADRE peptide was carried out as described in the paragraph immediately following the subheading "Site-specific modification of pyrroline-carboxy-lysine (PCL) containing proteins by various other molecules". Figure 50A is a MALDI-TOF mass spectrometric analysis of mTNF-Gln21PCL conjugated with PX2-PADRE (3465-143; see Example 38-11) at pH 5.0, and Figure 50B is mTNF conjugated with PX2-PADRE at pH 7.5. MALDI-TOF mass spectrometric analysis of -Q21PCL. The expected mass for the uncoupled protein is 19275 Da, and the expected mass for the coupled protein is 20842 Da. 50C is an ESI mass spectrometric analysis of mTNF-Gln21PCL conjugated with BHA-exPADRE (3647-104; see Example 38-10). At this time, the expected mass for the uncoupled protein is 19275 Da, and the expected mass for the coupled protein is 21317 Da. In addition, Figure 51 is an ESI mass spectrum showing the coupling of BHA-exPADRE to mEGF-Tyr10PCL (expected mass 7296 Da for uncoupled protein; expected mass 9338 Da for coupled protein).

Example 31 : Immunomodulator: Coupling of phospholipids

In order to confirm whether the immunomodulator and phospholipid can be coupled to a protein in which one or more PCL residues are incorporated therein, mEGF-Tyr10PCL (Example 12) was used using ABA phospholipid reagent (TU3627-092; Example 43-1). Reference) was conjugated with phospholipid (DOPE). Coupling of ABA-DOPE to mEGF-Tyr10PCL (MW = 7296 Da) was performed in 20 mM HEPES (pH 7.0) and 1% (v/v) DMSO at 25° C. for 16 hours. The conjugation reaction was initiated by adding 10 μM mEGF-Tyr10PCL and 100 μM ABA-DOPE. The formation of the protein conjugate was confirmed by electrospray ionization-mass spectrometry (ESI-MS) analysis of the conjugation of DOPE to mEGF-Y10PCL (FIG. 49B; expected mass for uncoupled protein = 7296; conjugation) Mass = 8227 Da).

Example 32 : Protein of Oligonucleotide: Coupling to CpG Peptide

To ensure that oligonucleotides and CpG immunomodulatory agents can be coupled to proteins in which one or more PCL residues are incorporated, CpG reagent BHA-BG1 (3647-057; see Examples 38-12) or CpG reagent BHA- BG2 (3597-167; see Examples 38-14) was used to conjugate mTNF-Gln21PCL and mEGF-Tyr10PCL (see Examples 11 and 12) with CpG oligonucleotides. Coupling of CpG oligonucleotides was carried out as described in the paragraph immediately following the subheading “Site-specific modification of pyrroline-carboxy-lysine (PCL) containing proteins by various other molecules”. The coupling of BHA-BG1 (7.4 kDa) and BHA-BG2 (7.4 kDa) to mTNF-Gln21PCL (19.3 kDa) was confirmed by gel shift assay (Figure 52A), and BHA to mEGF-Tyr10PCL (7.2 kDa). The coupling of -BG2 (7.4 kDa) was also confirmed by gel shift assay (Figure 52B).

Example 33: Synthesis of reactive pyrrolysine analogs

Example 33-1: (S)-2-amino-6-(3- oxobutanamido ) hexanoic acid Hydrochloride Synthesis of (TU3000-016)

According to the procedure described in the literature [Bing Hao et al., ChemBio 2004, 11, 1317-24] & its data, the removal of the Cbz group is carried out in the presence of HCl so that the HCl salt is obtained (S)-methyl 6-amino- 2-(2,2,2-trifluoroacetamido)hexanoate hydrochloride (TU2982-126) was prepared.

(S)-methyl 6-amino-2-(2,2,2-trifluoroacetamido)hexanoate hydrochloride (0.943 g, 3.22 mmol), N,N-diisopropyl in a 40 mL glass vial Diketone (0.37 mL) was added to ethylamine (DIEA, 1.39 mL) and dichloromethane (DCM, 10 mL) _{under N 2} atmosphere, and the reaction was stirred at ambient temperature for 16 hours. The reaction mixture was diluted with ethyl acetate (EtOA), washed successively with H ₂ O, 1 N HCl, H ₂ O, saturated aq Na ₂ CO ₃ , and saturated aq NaCl, _{dried over Na 2} SO ₄ , filtered and , Concentrated under reduced pressure. The residue _{was purified by SiO 2} flash chromatography, (S)-methyl-6-(3-oxobutanamido)-2-(2,2,2-trifluoroacetamido)hexanoate ( TU3000-012) was obtained as a yellow oil.

(S)-methyl 6-(3-oxobutanamido)-2-(2,2,2-trifluoroacetamido)hexanoate (0.736 g, 2.16 mmol) in 1 N aq NaOH (6.5 mL ) And 10 mL H ₂ O at ambient temperature for 18 hours. It was confirmed that the reaction was complete using LC-MS analysis. The reaction mixture was concentrated under reduced pressure. The residue was treated with an excess of 1 N aq HCl and concentrated to dryness under reduced pressure to give (S)-2-amino-6-(3-oxobutanamido)hexanoic acid hydrochloride (TU3000-016). I did.

Synthesis of (S) -5- (4- acetyl-benz amido) - 1-carboxy-pentane-1-aminium chloride fried (TU3000-004): Example 33-2

(S)-Methyl 6-amino-2-(2,2,2-trifluoroacetamido)hexanoate hydrochloride (TU2982-126) (303 mg, 1.03 mmol), 4-acetylbenzoic acid (190 mg ), HATU (418 mg), DIEA (523 μL) and DMF (8 mL) were combined and stirred at ambient temperature for 18 hours. The reaction mixture was diluted with EtOA, washed successively with H ₂ O, 1 N HCl, H ₂ O, saturated aq Na ₂ CO ₃ , and saturated aq NaCl, _{dried over Na 2} SO ₄ , filtered, and concentrated under reduced pressure. Made it. The residue _{was purified by SiO 2} flash chromatography to give (S)-methyl-6-amino-2-(2,2,2-trifluoroacetamido)hexanoate hydrochloride (TU2982-136). Obtained.

(S)-Methyl 6-(4-acetylbenzamido)-2-(2,2,2-trifluoroacetamido)hexanoate (TU2982-136) (0.473 g) was added to 1 in 10 mL MeOH Treated with Naq NaOH (2.36 mL) at ambient temperature for 18 hours. It was confirmed that the hydrolysis of the methyl ester was completed using LC-MS analysis, and that the trifluoroamide residue remained as it was. The reaction was heated at 60° C. for 5 hours, at which point it was confirmed by LC-MS analysis that the amide hydrolysis was almost complete. The reaction mixture was cooled and concentrated under reduced pressure. The residue was treated with an excess of 1 N aq HCl and concentrated to dryness under reduced pressure, ((S)-5-(4-acetylbenzamido)-1-carboxypentane-1-aminium chloride (TU3000- 004) was obtained as a pale yellow solid.

Example 33-3: ((S)-5-(5- acetylthiophene- 2 -carboxamido )-1- carboxypentane - 1-aminium chloride (TU3000-006), (S)-5- (3-acetylbenzamido)-1-carboxypentane-1-aminium chloride (TU3000-008), and (S)-5-(4-acetyl-1-methyl-1H-pyrrole-2-carboxami Figure) Synthesis of -1-carboxypentane-1-aminium chloride (TU3000-010)

(S)-5-(5-acetylthiophene-2-carboxamido)-1-carboxypentane-1-amido in the same manner as TU3000-004, except that the corresponding acid is used instead of 4-acetylbenzoic acid. Nium chloride (TU3000-006), (S)-5-(3-acetylbenzamido)-1-carboxypentane-1-aminium chloride (TU3000-008), and (S)-5-(4-acetyl -1-Methyl-1H-pyrrole-2-carboxamido)-1-carboxypentane-1-aminium chloride (TU3000-010) was prepared. The acids used were in the case of TU3000-006, TU3000-008, and TU3000-010, respectively, 4-acetyl-1-methyl-1H-pyrrole-2-carboxylic acid, 5-acetylthiophene-2-carboxylic acid and 3- It was acetylbenzoic acid.

Example 33-4: (S)-2-amino-6-(3- oxocyclobutanecarboxamido ) hexanoic acid (TU3205-030).

Iodomethane (2.0 mL), K ₂ CO ₃ (5.60 g), (S)-6-(benzyloxycarbonylamino)-2-(tert-butoxycarbonylamino)hexanoic acid (1) (Novabio Chem (NovaBiochem, A29340), and anhydrous DMF (20 mL) were combined and stirred at ambient temperature for 2 hours. The reaction mixture was subjected to aqueous workup to give (S)-methyl 6-(benzyloxycarbonylamino)-2-(tert-butoxycarbonylamino)hexanoate (TU3000-090) as a clear oil.

(S)-methyl 6-(benzyloxycarbonylamino)-2-(tert-butoxycarbonylamino)hexanoate (8.60 g) was added hydrogen 1 in 150 mL MeOH on 5% palladium on activated carbon (1.08 g) Hydrogenated for 3 hours at ambient temperature under atm. The spent catalyst was removed by passing through a pad of Celite by vacuum filtration and washed with MeOH. The combined filtrate and washings were concentrated under reduced pressure to give (S)-methyl 6-amino-2-(tert-butoxycarbonylamino)hexanoate (TU3000-128) as a clear viscous oil.

A 1 M stock solution of (S)-methyl 6-amino-2-(tert-butoxycarbonylamino)hexanoate in 20 mL DMF was prepared and used. 2 mL aliquots of (S)-methyl 6-amino-2-(tert-butoxycarbonylamino)hexanoate stock solution, 3-oxocyclobutanecarboxylic acid (2) (Parkway ^* BX -102, 283 mg), HATU (800 mg), DIEA (1.0 mL) and 8 mL DMF were combined in a 40 mL glass vial and stirred at ambient temperature overnight. The reaction was confirmed to be complete by LC-MS analysis. The reaction mixture was diluted with EtOAc, and water, saturated aq. Washed successively with NaCl, _{dried over MgSO 4} , filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash chromatography (hexane/EtOAc) to obtain (S)-methyl 2-(tert-butoxycarbonylamino)-6-(3-oxocyclobutanecarboxamido)hexanoate. (TU3000-140) was obtained as a clear viscous oil.

(S)-Methyl 2-(tert-butoxycarbonylamino)-6-(3-oxocyclobutanecarboxamido)hexanoate (0.501 g) in 20 mL 4 M HCl in 1,4-dioxane The furnace was treated at ambient temperature for 20 minutes, and the solvent was removed under reduced pressure. The resulting viscous oil _{was dissolved in 10 mL CH 3} CN, and a small amount of crystals of the title compound prepared on a small scale were seeded. The resulting crystals were collected by vacuum filtration, washed with CH ₃ CN and dried under reduced pressure, (S)-methyl 2-amino-6-(3-oxocyclobutanecarboxamido)hexanoate (TU3205 -016) was obtained as a colorless solid.

(S)-Methyl 2-amino-6-(3-oxocyclobutanecarboxamido)hexanoate (0.297 g) and 18 mL H ₂ O were placed in a 40 mL glass vial. 2.2 mL 1 N NH ₄ OH was added to the resulting clear solution, and the reaction was shaken at ambient temperature for 22 hours, at which point the reaction was confirmed to be complete by LCMS analysis. The reaction mixture was frozen and lyophilized to give (S)-2-amino-6-(3-oxocyclobutanecarboxamido)hexanoic acid (TU3205-030) as colorless crystals.

Example 34: Synthesis of reactive pyrrolysine intermediate

Example 34-1: Synthesis of lithium 2- (4-acetyl-3-aminophenoxy) acetate (TU3205 -042)

1-(4-hydroxy-2-nitrophenyl)ethanone (Carbocore, 181 mg, 1.00 mmol), ethyl 2-bromoacetate (183 mg, 1.10 mmol), potassium carbonate (138 mg. 1.00 mmol) and DMF (5 mL) were combined in a 20 mL glass vial and stirred at 60° C. for 2 hours, at which point LC-MS analysis showed the reaction to be complete. The reaction mixture was diluted with water, extracted with EtOAc, and washed with saturated NaCl aq. 1-(4-hydroxy-2-nitrophenyl)ethanone (0.802 g, 4.43 mmol), ethyl 2-bromoacetate (0.813 g, 4.87 mmol), potassium carbonate (0.612 g. 4.43 mmol) and DMF (25 mL) was used to repeat the reaction, followed by work-up in the same manner. The combined EtOAc extracts were dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure to give ethyl 2-(4-acetyl-3-nitrophenoxy)acetate (TU3205-034) as a dark yellow oil.

Ethyl 2-(4-acetyl-3-nitrophenoxy)acetate (TU3205-034) (111 mg) in MeOH (5 mL) was added to 10% palladium on charcoal (11 mg) at ambient temperature under _{H 2 atm.} Hydrogenated. After 30 minutes, the reaction was confirmed to be complete by LC-MS analysis. The reaction was repeated with TU3205-034 (1.45 g), 10% palladium on charcoal (140 mg), and MeOH (80 mL). The two reaction mixtures were combined and the spent catalyst was removed through a pad of celite by filtration. The filtrate was concentrated under reduced pressure to give ethyl 2-(4-acetyl-3-aminophenoxy)acetate (TU3205-036) as a dark yellow oil.

Ethyl 2-(4-acetyl-3-aminophenoxy)acetate (TU3205-036) (1.02 g) was dissolved in THF (17 mL) and aqueous LiOH (1 M, 4.3 mL) at ambient temperature for 1 hour. Processed. LC-MS analysis indicated that the reaction was complete. The reaction mixture was concentrated under reduced pressure to give lithium 2-(4-acetyl-3-aminophenoxy)acetate (TU3205-042) as a yellowish solid.

Example 34-2: Synthesis of lithium 2- (3-amino-4-formyl-phenoxy) acetate (TU3627 -018).

While cooling in an ice bath, borontribromide (24 g) was added dropwise to 4-methoxy-2-nitrobenzaldehyde (Carbocore, CO-0119, 5.5 g) and 200 mL DCM in a 500 mL round bottom flask. The reaction was stirred at the same temperature for 30 minutes and then at ambient temperature for 3 hours. The reaction mixture was carefully poured into ice water and allowed to stand at ambient temperature for 3 days. The aqueous mixture was extracted with EtOAc and saturated aq. Washed with NaCl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. _{The crude was purified by SiO 2} gel flash chromatography using a linear gradient of 20 to 60% EtOAc in hexane (Rf. 0.32, 50% EtOAc in hexane) to give TU3627-002 as orange crystals.

TU3627-002 (1.87 g), ethyl bromoacetate (Aldrich, 1.5 mL), potassium carbonate (2.32 g) and DMF were combined and stirred at ambient temperature for 18 hours. The reaction mixture was partitioned between EtOAc and water. The organic layer was separated, and saturated aq. Washed with NaCl, _{dried over MgSO 4} , filtered and concentrated under reduced pressure. _{The crude was purified by SiO 2} gel flash chromatography using a linear gradient from 5 to 35% EtOAc in hexanes (Rf. 0.32, 35% EtOAc in hexanes) to give TU3627-008 as a dark yellow oil.

TU3627-008 was reduced using the method described in Merlic et al., J. Org. Chem., 1995, 60, 3365-69. Specifically, TU3627-008 (1.717 g), iron powder (3.79 g), EtOH (45 mL), H ₂ O (11 mL) and concentrated HCl (180 μL) were combined and heated to reflux for 2 hours. The reaction mixture was filtered and the filtrate was concentrated. The residue was taken on silica gel using a linear gradient from 5 to 60% solvent B in solvent A (solvent A: NEt ₃ in 5% hexane; solvent B: NEt ₃ in 5% EtOAc) (Rf. 0.34, 35% EtOAc in hexane). Purification by flash chromatography gave TU3627-014 as light yellow crystals.

TU3627-014 (0.608 g) in 5 mL THF was added to 1 M aq. Treated at ambient temperature with 2.72 mL aliquots of LiOH. After 20 minutes, it was confirmed that the reaction was complete by LC-MS analysis. The reaction mixture was concentrated to dryness under reduced pressure to give lithium 2-(3-amino-4-formylphenoxy)acetate (TU3627-018) as a yellow solid.

Example 34-3: Synthesis of lithium 4-(3-acetyl-4- aminophenoxy ) butanoate ( TU3627 -064) .

A mixture of 4'-chloro-2'-nitroacetophenone (Bionet cat# 3W0333, 1.00 g), potassium acetate (4.91 g) and DMSO was heated at 170° C. for 20 minutes under microwave irradiation. The reaction mixture was partitioned between EtOAc and water. The organic layer was separated, and saturated aq. Washed with NaCl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Another reaction was carried out in the same way, and the crude products from the two reactions were combined and purified by silica gel flash chromatography (EtOAc). The title compound was obtained as an orange solid.

A mixture of 4'-hydroxy-2'-nitroacetophenone (1.12 g), ethyl 4-bromobutanoate (1.33 g), potassium carbonate (0.94 g) and 4 mL DMF was stirred at 60° C. for 6.5 hours I did. The reaction mixture was partitioned between EtOAc and water. The organic layer was separated, and saturated aq. Washed with NaCl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by silica gel flash chromatography (hexane/EtOAc) to give the title compound 1 as yellow crystals.

TU633-148 (1.48 g) was reduced by the method described by Merrick, Camerlic et al., J. Org. Chem., 1995, 60, 3365-69, as described herein. TU3627-056 was obtained after flash chromatography on silica gel using a linear gradient of 5→60% solvent B in solvent A (solvent A: NEt ₃ in 5% hexane; solvent B: NEt _{3 in 5% EtOAc).} I did.

TU3627-056 (0.50 g) in 3 mL THF was added to 1 M aq. A 1.9 mL aliquot of LiOH was treated at ambient temperature for 18 hours. The reaction was confirmed to be complete by LC-MS analysis. The reaction mixture was concentrated to dryness under reduced pressure to give lithium 4-(3-acetyl-4-aminophenoxy)butanoate (TU3627-064) as a yellow solid.

Example 34-4: lithium 4- (3-amino-4-formyl-phenoxy) butanoate: Synthesis of (TU3627 -074).

A mixture of 2-nitro-4-hydroxybenzaldehyde (2.85 g), ethyl 4-bromobutanoate (3.66 g), potassium carbonate (2.84 g) and DMF (20 mL) was stirred at ambient temperature for 50 hours. . The reaction mixture was _{partitioned between H 2} O and EtOAc. The organic layer was separated and washed with saturated NaHCO ₃ aq. The combined aqueous layer was extracted with EtOAc. The combined organic layers were washed with light citric acid, saturated NaCl aq, _{dried over MgSO 4} , filtered and concentrated. The residue was purified by silica gel flash chromatography using a linear gradient of 20 to 40% EtOAc in hexanes (Rf: 0.35, 35% EtOAc in hexanes) to give the title compound as a yellow oil.

TU3627-062 (3.89 g) was reduced by the method described in Merrick's document Camerlic et al., J. Org. Chem., 1995, 60, 3365-69, as described herein. TU3627-066 was obtained as a yellow solid after silica gel flash chromatography using a linear gradient of solvent B in 20 to 60% solvent A (solvent A: NEt ₃ in 5% hexane; solvent B: NEt _{3 in 5% EtOAc).} . Rf: 0.51, 50% EtOAc in hexane.

TU3627-066 (1.00 g) in 6 mL THF was treated with 3.98 mL of 1 M aqueous LiOH at ambient temperature for 4 hours. Most of the solvent was removed under reduced pressure, and the resulting cloudy mixture was diluted with double deionized water, freeze and lyophilized to obtain lithium 4-(3-amino-4-formylphenoxy)butanoate (TU3627- 074) was obtained as a pale yellow solid.

Example 34-5: Synthesis of lithium 3-(3-acetyl-4 -aminophenyl ) propanoate ( X3547-1 ) .

1-(2-amino-5-bromophenyl)ethanone (642 mg), Pd(OAc) ₂ (33.7 mg), and P( o Tolyl) ₃ (137 mg) in anhydrous DMF (10 mL) in a pressure tube ) Methyl acrylate (351 μl) and TEA (1.4 mL) were added to the mixture. The mixture _{was flushed with N 2} for 3 minutes, then sealed and heated at 110° C. for 4 hours. The reaction mixture was cooled to ambient temperature and then partitioned between ethyl acetate and water. The aqueous layer was extracted once with ethyl acetate, and the combined organic layers were washed with brine, dried (Na ₂ SO ₄ ), filtered and the solvent was removed under vacuum. The crude residue was purified by silica gel flash chromatography (EtOAc/hexane) to give the product.

(E)-Methyl 3-(3-acetyl-4-aminophenyl)acrylate (219 mg) was reduced at room temperature with 5% Pd/C (21.9 mg) in MeOH with a hydrogen balloon, after filtration and concentration The product (quantitative) was obtained. The product was used in the next step without further purification.

Methyl 3-(3-acetyl-4-aminophenyl)propanoate (221 mg) and 4 M LiOH (0.275 mL) were added to 3 mL THF/water (v/v = 3/1). After the reaction was completed, the solvent was removed under reduced pressure to obtain lithium 3-(3-acetyl-4-aminophenyl)propanoate (X3547-1).

Example 34-6: Synthesis of lithium 3-(4-acetyl-3 -aminophenyl ) propanoate ( X3547-8 ) .

CH ₃ CN solution (10 mL) containing 4-bromo-3-nitrobenzaldehyde (1.150 g) while stirring a solution of ethyl triphenylphosphoranilidine acetate (1.742 g) dissolved in CH _{3 CN (15 mL)} Was added to. The reaction mixture was refluxed overnight. After cooling the mixture, the solvent was removed under reduced pressure to give a crude solid. The pure product X3471-146 was obtained as a white solid after silica gel flash column chromatography (hexane/EtOAc, 9:1).

The product X3471-146 (632 mg) and PdCl ₂ (PPh ₃ ) ₂ (148 mg) from the above step were dissolved in DMF (5.0 mL) under _{N 2 in a Schlenk tube.} Tributyl(1-ethoxyvinyl)stannan (711 μl) was added with stirring. The mixture was heated at 100° C. overnight. After cooling the mixture, the solvent was removed under reduced pressure, diluted with DCM and washed with water and brine. After removal of DCM, the residue was purified by silica gel flash column chromatography (15%-25% EtOAc in hexane) to give compound X3471-154.

Compound X3471-154A was treated with 20 mL 1 N HCl at room temperature for 4 hours. Removal of the solvent gave (E)-ethyl 3-(4-acetyl-3-nitrophenyl)acrylate X3471-154B.

Compound X3471-154B (263 mg) to ethyl 3-(4-acetyl-3-aminophenyl)propanoate X3547-2 at 1 atm under hydrogen using 5% Pd/C (26 mg) in 5.0 mL MeOH. Reduced.

Compound X3547-2 was treated at room temperature using a mixture of 4 M LiOH (0.248 mL), THF (3.0 mL) and water (1.0 mL). After the reaction was completed, the reaction mixture was lyophilized to obtain lithium 3-(4-acetyl-3-aminophenyl)propanoate (X3547-8).

Example 34-7: Lithium 5- (3-amino-4-formylphenyl) pentanoate synthesis of (X3547 -30).

4-bromo-2-nitrobenzaldehyde (460 mg), Pd(PPh ₃ ) ₂ Cl ₂ (70 mg), and CuI (38 mg), methyl pent-4-inoate (269 mg) in TEA (5.0 mL) ) Was stirred at room temperature until the reaction was complete. The crude residue was purified by silica gel flash chromatography (15% EtOAc in hexane) to give methyl 5-(4-formyl-3-nitrophenyl)pent-4-inoate.

Methyl 5-(4-formyl-3-nitrophenyl)pent-4-inoate (522 mg) was reduced at room temperature with 5% Pd/C in MeOH (53 mg) with a hydrogen balloon. After filtration, it was concentrated under reduced pressure to obtain methyl 5-(3-amino-4-formylphenyl)pentanoate.

Methyl 5-(3-amino-4-formylphenyl)pentanoate (447 mg) was treated with LiOH (88 mg) in 8.0 mL THF/water (v/v = 3/1). After the reaction was completed, the solvent was removed to obtain lithium 5-(3-amino-4-formylphenyl)pentanoate (X3547-30).

Example 34-8: Synthesis of 3-(4-acetyl-3 -aminophenyl )-2 -aminopropanoic acid ( X3179-96 ) .

Sodium hydride (60% in mineral oil, 1.74 g) was washed with hexane and dispersed in DMF (12 ml). Diethyl acetamidomalonate (10.4 g) in DMF (30 ml) and 4-(bromomethyl)-1-fluoro-2-nitrobenzene (10.2 g) in DMF (10 ml) are added successively, After the reaction mixture was stirred at room temperature for 4 hours, the solvent was removed under reduced pressure. The residue was purified by silica gel flash chromatography (10-20% EtOAc in DCM) to give diethyl 2-acetamido-2-(4-fluoro-3-nitrobenzyl)malonate.

To diethyl 2-acetamido-2-(4-fluoro-3-nitrobenzyl)malonate (7.41 g) and nitroethane (6.0 mL) in EtOAc was added DBU (9.0 mL) and the mixture was at room temperature Stir for 16 hours. The reaction mixture was concentrated and the residue was dissolved in methanol (35 mL). Aqueous H ₂ O ₂ (30%, 10.2 mL) and 10% aqueous NaHCO ₃ (10.2 mL) were added and the mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc, washed with 1 N HCl, brine, _{dried over MgSO 4} , filtered and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (10-20% EtOAc in DCM) to give diethyl 2-acetamido-2-(4-acetyl-3-nitrobenzyl)malonate.

Diethyl 2-acetamido-2-(4-acetyl-3-nitrobenzyl)malonate (2.0 g) was dissolved in 10 mL 37% HCl, heated at 100° C. overnight and cooled. The resulting solid was collected by filtration to give 3-(4-acetyl-3-nitrophenyl)-2-aminopropanoic acid.

3-(4-acetyl-3-nitrophenyl)-2-aminopropanoic acid was reduced under _{1 atm H 2 with 5% Pd on carbon in MeOH.} After filtration, it was concentrated under reduced pressure to give 3-(4-acetyl-3-aminophenyl)-2-aminopropanoic acid (X3179-96).

Example 34-9: 1-(2-amino-5- Bromophenyl ) Ethanone ( X1436 -132).

A flask filled with 1-(2-aminophenyl)ethanone (135 mg) and DCM (2.5 mL) was cooled to -10° C. and NBS (178 mg) was added in several portions over 30 minutes. The reaction mixture was diluted with 10 mL DCM, washed with saturated aqueous NaHCO ₃ , dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give the title compound.

Example 34-10: 1-(2-amino-5- Iodophenyl ) Ethanone ( X1436 -134).

A flask filled with 1-(2-aminophenyl)ethanone (405 mg) and DCM (20 mL) was cooled in an ice/water bath and NIS (675 mg) was added in 3 portions. The reaction was stirred at 0 °C. The reaction mixture was diluted with 30 mL DCM, washed with saturated aqueous NaHCO ₃ , dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude material was purified by preparative RP-HPLC to give the title compound.

Example 34-11: 2-amino-4- Bromobenzaldehyde ( X3547 -158).

Iron powder (1.20 g), water (2.0 mL), and HCl (12 N, 40 μl) were added successively to a solution of 2-nitrobenzaldehyde (230 mg) in ethanol (8.0 mL). After stirring at 95° C. for 90 minutes, the reaction mixture was filtered at high temperature. After washing with ethanol, the filtrates were combined and the solvent was removed under vacuum. The crude was purified by silica gel flash chromatography (40:55:5 hexanedecyl acetate/triethylamine) to give 2-amino-4-bromobenzaldehyde as yellow crystals.

Example 35: PCL And synthesis of pyrrolysine biosynthetic precursors

Example 35-1: ammonium DL -One- Pyrroline -5- Carboxylate Synthesis of (3793-007).

The HCl salt of H-DL-δ-DL-hydroxyLys-OH (118 mg, 0.5 mmol) was applied to a cation exchange SPE cartridge and the amino acids were eluted with ammonium hydroxide. NaIO ₄ (107 mg) was added to the ammonium eluent and the reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was lyophilized and the crude product was acidified to pH 2.6 with HCl and purified by cation exchange SPE. After decanting the first 10 mL of H ₂ O eluent, the next 30 ml of H ₂ O eluent was collected and _{neutralized immediately with 1 N NH 4} OH _{( aq )} (0.5 mL). After lyophilization, the desired product was obtained as a light yellow powder.

Example 35-2: H-L- Lys -N ^ε -(D- Orn )- OH Synthesis of (3793-031)

Boc-D-Orn(Boc)-OH (1.097 g) was treated with HATU (1.255 g) and DIEA (1.53 mL) in DMF (5 mL) for 1 hour. Then, a DMF solution (5 mL) and DIEA (627 μL) of Boc-Lys-OMe (acetate salt, 1.057 g) were added to the reaction mixture, and the reaction was stirred at room temperature for 2 hours. The reaction mixture _{was partitioned between 10% NaCl ( aq )} (30 mL) and EtOAc (60 mL). The EtOAc layer was washed with 5% citric acid (30 mL), 10% NaHCO _3(aq) (30 mL), and brine (30 mL). The EtOAc layer was _{dried over Na 2} SO _{4 (s)} , filtered and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography using an 80 g silica column and gradient elution of 0-15% MeOH/DCM, Boc-Lys(Boc-D-Orn(Boc))-OMe (3793-026) Was obtained as a colorless oil.

To 65% aqueous acetonitrile solution (5 mL) of Boc-Lys(Boc-D-Orn(Boc))-OMe (214 mg) was added concentrated HCl (1 mL), and the reaction mixture was stirred vigorously for 2 hours. The reaction mixture was lyophilized and purified with 1 N NH ₄ OH in _{20% MeCN ( aq )} by cation exchange chromatography using an SCX SPE cartridge. The methyl ester was hydrolyzed in NH ₄ OH eluent overnight. After lyophilization, HL-Lys-N ^ε -(D-Orn)-OH (3793-031) was obtained as a white powder.

Example 35-3: N-((6 -chloropyridin- 3-yl) methyl )-1 -pyrroline -5 -carboxamide Synthesis of (3647-061).

T-butyl pyrocarbonate (6.55 g) in dioxane (10 mL) was added dropwise to DL-δ-DL-hydroxyLys (1.99 g) in _{1 N NaOH ( aq ) (50 mL) and the reaction was brought to room temperature.} Stir overnight at. The reaction mixture was acidified to pH 3.0 with 1 N HCl _{( aq )} and extracted twice with EtOAc (200 mL). The EtOAc layers were combined, _{dried over Na 2} SO _{4 (s)} , filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel flash column chromatography with gradient elution of 0-10% MeOH/DCM to give Boc-DL-δ-DL-hydroxyLys(Boc) (3597-109) as a white solid.

Boc-DL-δ-DL-hydroxyLys(Boc) (435 mg) was treated with HATU (456 mg) and DIEA (523 μL) in 3.5 mL DMF for 1 hour. The resulting solution was added to 6-chloropyridin-3-ylmethylamine (143 mg) in 1.5 mL DMF solution at room temperature and the reaction was stirred for 2 days. The reaction mixture _{was extracted with 10% NaCl ( aq )} (15 mL) and EtOAc (30 mL). The EtOAc layer was washed with 5% citric acid (15 mL), 10% NaHCO ₃ (15 mL), and brine (15 mL). The EtOAc layer was then _{dried over Na 2} SO _{4 (s)} , filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel flash column chromatography with gradient elution of 0-15% MeOH/DCM to give the desired product (3647-016) as a white solid.

The above product (3647-016) (263 mg) was stirred overnight at room temperature in a solution of _{Et 2} O (7 mL) and 1 N HCl _{( aq ) (7 mL).} After the reaction mixture was evaporated and lyophilized, the residue was purified by a cation exchange SPE cartridge to give the desired product (3647-037) as a white solid.

The above product (3647-037) (69.6 mg) _{was dissolved in 2 mL H 2} O (2 mL), and the pH was adjusted to 12 using _{1 N NaOH (aq).} PL-IO ₄ resin (Varian, 381 mg, 0.48 mmol) was added and the reaction was stirred at room temperature for 2 hours. The resin was removed by filtration and half of the filtrate was purified by preparative HPLC to give the desired product (3647-061) as a white solid.

Example 36: PCL Synthesis of model compounds

Example 36-1: H-L- Lys -Nε-( DL -One- Pyrroline -5-carbonyl)- OH Synthesis of (3647-125).

Boc-DL-δ-DL-hydroxyLys(Boc) (3597-109) (886 mg) was activated by HATU (932 mg) in 3.0 mL DMF in the presence of DIEA (1068 μl) for 1 hour and 3.0 Added to Boc-Lys-OMe (549 mg) in mL DMF (3.0 mL). The reaction was stirred at room temperature for 36 hours. The reaction mixture _{was partitioned between 10% NaCl ( aq )} (30 mL) and EtOAc (60 mL). The EtOAc layer was washed with 5% citric acid (15 mL), 10% NaHCO ₃ (15 mL), and brine (15 mL). The EtOAc layer was _{dried over Na 2} SO _{4 (s)} , filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel flash column chromatography using 0-15% MeOH/DCM, and then purified by RP-C18 SPE using elution of _{25% and 65% MeCN ( aq ), and the desired product (3647} -099) was obtained as a white solid.

To Boc-Lys(Boc-DL-δ-DL-hydroxyLys(Boc))-OMe (3647-099) (84.1 mg) in 1 mL Et _{2 O was added 4 N HCl ( aq )} (3 mL) and , The reaction was stirred at room temperature for 3 hours. Removal of the solvent gave 61.1 mg of crude product (3647-120) as a white solid.

The above crude product (3647-120) (61.1 mg) _{was dissolved in 1 mL H 2} O, and the pH was adjusted to 12 using _{1 N NaOH (aq).} The mixture was stirred at room temperature for 1 hour to hydrolyze the methyl ester. Then NaIO ₄ (29.9 mg) was added to the reaction mixture, and the reaction was stirred at room temperature for an additional 15 minutes. The reaction mixture _{was neutralized with 1 N HCl (aq)} and loaded into cation exchange SPE for purification to obtain HL-Lys-Nε-(DL-1-pyrroline-5-carbonyl)-OH (3647-125). Obtained.

Example 36-2: H-L- Lys -N ^ε -( DL -One- Pyrroline -2-carbonyl)- OH Synthesis of (3793-011).

Sodium 1-pyrroline-2-carboxylate (3647-164) was prepared using the following method described in the literature [Gyoergy Szoelloesi et al., Chirality, 2001, 13(10), 619-624]. .

Triethylamine (8.4 mL) was added to H-Pro-OMe HCl (7.512 g) in ether (27 mL) and the reaction was stirred for 2 hours. The reaction mixture was filtered and Et ₂ O was removed by evaporation. The residue was purified by vacuum distillation to give 4.119 g of H-Pro-OMe as a colorless oil. _{t-BuOCl (3.59 mL) was added dropwise to a solution of Et 2} O (100 mL) in H-Pro-OMe (4.089 g), and the reaction was stirred at -50°C for 1 hour. The reaction mixture was warmed to room temperature and triethylamine (4.64 mL) was added to the reaction mixture, followed by stirring for 3 days. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by vacuum distillation to give methyl pyrroline-2-carboxylate (3647-154) as a colorless oil.

Methyl pyrroline-2-carboxylate (2.265 g _{) was dissolved in 1 N NaOH ( aq )} (17.8 mL) and stirred at room temperature for 1 hour. The reaction mixture was lyophilized to give crude sodium pyrroline-2-carboxylate (3647-164) as a white powder.

Sodium 1-pyrroline-2-carboxylate (459 mg) and DPPA (886 μL) were added to Boc-Lys-OMe (acetate salt, 897 mg) and DIEA (1184 μL) in 20 mL DMF and the reaction was Stirred at room temperature for 24 hours under N _2. The reaction mixture _{was partitioned between 10% NaCl ( aq )} (50 mL) and EtOAc (100 mL). The EtOAc layer was _{dried over Na 2} SO _{4 (s)} , filtered and concentrated under reduced pressure to give the crude product as a light orange oil. The crude product was purified by silica gel flash column chromatography with gradient elution of 0-15% MeOH/DCM and then purified by RP-C18 SPE to give the desired product (3647-167) as a colorless oil.

HCl (conc., 1 mL) was added to 50% MeCN(aq) of the product (3647-167) (49.3 mg) It was added to the solution (5 mL) and the reaction was stirred at room temperature for 2 hours. After lyophilization, the residue was purified by a cation exchange SPE cartridge. The methyl ester was hydrolyzed overnight with _{NH 4} OH _{( aq ) in the eluent.} After lyophilization, the desired product (3793-011) was obtained as a light yellow powder.

Example 37: 2- AAP - PEG Synthesis of reagents

Synthesis of other 2-AAP-PEG derivatives used in the examples provided herein is described below. The 2-amino-acetophenone derivatives of PEG with molecular weights of approximately 500, 2400, 23000 Da as well as 5000 and 10000 Da were synthesized as follows:

Example 37-1: TU3205 -044 (0.5 kDa 2- AAP - mPEG ) Of the synthesis

Lithium 2-(4-acetyl-3-aminophenoxy)acetate (TU3205-042) (55.9 mg) was charged to a 10 mL round bottom flask, and DMF (3 mL) and HATU (98.9 mg) were added. The resulting slurry was stirred at ambient temperature for 40 minutes. During this period the reaction turned into a yellow solution. Then to the reaction was added mPEG-amine (Quanta Biodesign, MW 383.5, 100 mg) in 2 mL DMF. LC-MS analysis after 5 minutes indicated the reaction was complete. The reaction mixture was stirred for an additional 40 minutes and concentrated under reduced pressure. The residue was purified by SiO ₂ flash chromatography (MeOH in 3% DCM) to give TU3205-044 (0.5 kDa 2-AAP-mPEG) as a yellow viscous oil.

Example 37-2: TU3205 -048 (2.4 kDa 2- AAP - PEG ) Of the synthesis

Lithium 2-(4-acetyl-3-aminophenoxy)acetate (TU3205-042) (12.2 mg) was charged to a 10 mL round bottom flask, and DMF (1 mL) and HATU (21.5 mg) were added. The resulting slurry was stirred at ambient temperature for 35 minutes. During this period the reaction turned into a yellow solution. Then to the reaction was added mPEG-amine (Quanta Biodesign, MW 2209, 100 mg) in 2 mL DMF. The reaction mixture was stirred for 18 hours and concentrated under reduced pressure. The residue was purified by SiO ₂ flash chromatography (MeOH in DCM) to give TU3205-048 (2.4 kDa 2-AAP-mPEG) as a yellow viscous oil.

Example 37-3: TU3205 -052 (23 kDa 2- AAP - PEG ) Of the synthesis

Lithium 2-(4-acetyl-3-aminophenoxy)acetate (TU3205-042) (21.5 mg) and HATU (38.0 mg) were charged to a 10 mL round bottom flask, and DMF (0.5 mL) was added. The resulting slurry was stirred at ambient temperature for 50 minutes. The resulting yellow solution was added to mPEG-NH ₂ (Laysan Bio, average MW 23k, average n = 520, 0.50 g) in 5 mL DMF in a 20 mL glass vial. The reaction was shaken at ambient temperature for 2.5 hours. The reaction mixture was then diluted with 10 mL water, a 2.5 mL aliquot of each solution was applied to a PD-10 column (GE Healthtech), and the desired product was eluted with water according to the supplier's instructions. The pooled aqueous solution was pooled, frozen and lyophilized to give TU3205-052 (23 kDa 2-AAP-mPEG) as a white solid. (Note; PEG reagent characterized by high MW was obtained only after it was conjugated to PCL containing protein.)

및

And

Were prepared in a similar manner to TU3205-052 using the corresponding 10,000 (average n = 225) and 5,000 (average n = 111) MW mPEG-NH _2. (Note; PEG reagent characterized by high MW was obtained only after it was conjugated to PCL containing protein.)

Example 37-4: TU633 -010: 2- Organoleptic Linker synthesis

Lithium 2-(4-acetyl-3-aminophenoxy)acetate (TU3205-042) (94.6 mg) and HATU (167 mg) were charged to a 10 mL round bottom flask, and DMF (2 mL) was added. The resulting slurry was stirred at ambient temperature for 45 minutes. To the resulting yellow solution was added 4,7,10-trioxa-1,13-tridecanediamine (Fluka, 44 mg) in 1 mL DMF. The reaction mixture was stirred at ambient temperature for 18 hours and concentrated under reduced pressure. The residue was purified by SiO ₂ flash chromatography (MeOH in DCM) followed by preparative reverse phase LC purification. The LC purified material was dissolved in EtOAc _{and treated with saturated aqueous NaHCO 3} to remove trifluoroacetic acid. Evaporation of the solvent gave the bifunctional linker TU633-010 as a clear oil.

Example 37-5: m- PEG - AAP 29k ( TU633 -084).

Lithium 2-(4-acetyl-3-aminophenoxy)acetate (187 mg) and HBTU (330 mg) were placed in a 20 mL glass vial, and 10 mL anhydrous DMF was added. The resulting slurry was stirred at ambient temperature. The reaction turned into a yellow solution within 20 minutes, and after 80 minutes a 9.5 mL aliquot of the reaction mixture was added to mPEG-NH ₂ (Raysan Bio, MW 28,700) dissolved in 40 mL of anhydrous DMF in a 100 mL round bottom flask. I did. The reaction was shaken at ambient temperature for 19 hours. The reaction mixture was then applied to 24 pieces of PD-10 column (GE Healthtech) and the desired product was eluted with water according to the supplier's instructions. The pooled eluent was frozen and lyophilized to obtain a white solid. The solid was dissolved in double deionized water and thoroughly dialyzed against double deionized water using a dialysis membrane of MWCO 3500. The dialyzed solution was frozen and lyophilized to give TU633-084 as a white fluffy solid. (Note; PEG reagent characterized by high MW was obtained only after it was conjugated to PCL containing protein.)

Example 37-6: mPEG - AAP -30k ( TU633 -120)

Lithium 3-(3-acetyl-4-aminophenyl)propanoate (139 mg) and HBTU (247 mg) were placed in a 20 mL glass vial, and 13 mL anhydrous DMF was added. The resulting slurry was stirred at ambient temperature for 30 minutes, and the resulting solution was used to prepare TU633-120, TU633-122, TU633-124 and TU633-126. In a 45 mL glass vial, mPEGamine (NOF Corp., SUNBRIGHT MEPA-30T MW 30,298, 3.0 g) was added, 20 mL of anhydrous DMF was added, and mPEGamine was dissolved in DMF by heating lightly. A 2.2 mL aliquot of the activated ester solution was added to the resulting mPEGamine solution, and the vial was shaken at ambient temperature for 18 hours and then at 37° C. for 24 hours. The reaction mixture was transferred to dialysis membrane tubing (Fisher Scientific, cat # 21-152-9, MWCO 3500) and thoroughly dialyzed against double deionized water over 2 days. The dialyzed solution was frozen and lyophilized to give TU633-120 as a white cotton-like solid. (Note; PEG reagent characterized by high MW was obtained only after it was conjugated to PCL containing protein.)

Instead of SUNBRIGHT MEPA-30T, NOF Corp. In a similar manner, except for using the corresponding activated PEG from the company, SUNBRIGHTMEPA-40T (MW 42036), SUNBRIGHT GL2-400PA (MW 42348), SUNBRIGHT GL4-400PA respectively.

및

And

을 제조하였다.

Was prepared.

(Note; PEG reagent characterized by high MW was obtained only after it was conjugated to PCL containing protein.)

Example 37-7: mPEG - ABA -30 kD ( TU3627 -024).

The title compound was in the same manner as TU633-120, except that lithium 2-(3-amino-4-formylphenoxy)acetate was used instead of lithium 3-(3-acetyl-4-aminophenyl)propanoate. Was prepared. (Note: The PEG reagent, characterized by high MW, was obtained only after it was conjugated to the PCL containing protein.)

Example 37-8: mPEG - ABA -30k ( TU3627 -084).

The title compound is the same as TU633-120, except that lithium 4-(3-amino-4-formylphenoxy)butanoate is used instead of lithium 3-(3-acetyl-4-aminophenyl)propanoate. Prepared in the same way. H NMR analysis confirmed that no unmodified starting PEG was present in the product. End point activity: greater than 95% by H-NMR.

Example 37-9: mPEG - ABA -40k ( TU3627 -086).

The title compound was prepared in the same manner as TU3627-084, except that SUNBRIGHTMEPA-40T was used instead of SUNBRIGHTMEPA-30T. H NMR analysis confirmed that no unmodified starting PEG was present in the product. End point activity: greater than 95% by H-NMR.

Example ^{37-10: N 1 - (18- (} 3- amino-4-formyl-phenoxy) -15- oxo -4,7,10- tree-oxa-14-aza-octadecyl) -N ¹⁹ - (18 -(3-amino-4-formylphenoxy)-15-oxo-4,7,10-trioxa-9,14-diazaoctadecyl)-4,7,10,13,16-pentaoxanona Synthesis of decane-1,19-diamide (X3678-40)

Lithium 4-(3-amino-4-formylphenoxy)butanoate (94.5 mg) was activated with HBTU (151.7 mg) in 4 mL DMF. Diamido-dPEG™ ₁₁ -diamine (Quanta Biodesign, Cat# 10361, 74.3 mg) was added to the reaction mixture and stirred at room temperature overnight. DIEA (17.5 μl, 0.1 mmol) was added and the mixture was heated at 40° C. for several hours. The title compound was isolated by preparative RP-HPLC and TFA was removed by passing it through a _{PL-HCO 3 MP SPE cartridge (Varian Inc.).}

Example 38: immune modulators to proteins Coupling Synthesis of reagents for

Example 38-1: N- (1- (3- amino-4-formyl-phenoxy) -2-oxo -7,10,13- tree oxa-3-aza-hexadecane -16- yl) -4- ((4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)methyl)benzamide (TU3627-042) synthesis.

T-Butyl 3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propylcarbamate (Quantabiodesign, cat # 10225, 500 mg) and DIEA (348 μl) in 10 mL DCM ) Was added 4-chloromethylbenzoyl chloride in 2 mL DCM while cooling in an ice bath. The reaction was stirred at the same temperature for 1 hour. The reaction mixture was diluted with EtOAc, washed _{successively with H 2} O, mild aqueous citric acid, aqueous NaHCO ₃ and saturated aqueous NaCl, _{dried over Na 2} SO ₄ , filtered, and concentrated under reduced pressure to make the product (A) clear. Obtained as a viscous oil.

The above product A (303 mg), compound B (200 mg), cesium carbonate (208 mg) and anhydrous DMSO were combined and the reaction was stirred at ambient temperature. After 18 hours, an additional 60 mg of compound A dissolved in 8 mL DMSO and an additional 30 mg of cesium carbonate were added to the reaction, and the reaction was stirred for 40 hours at ambient temperature, at which time compound B was only traced. Only the amount remained (confirmed by LCMS analysis). The reaction mixture was diluted with EtOAc, washed _{successively with H 2} O, saturated aqueous NaCl, _{dried over Na 2} SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography using a linear gradient of B in 1→5% solvent A (A: DMC; B: NH _{3 in 2% MeOH) to give the desired compound partially purified.} After recrystallization from MTBE, the product was obtained as light yellow crystals.

The Boc group of compound C was removed by treatment with 3 M methanolic HCl at ambient temperature. The reaction mixture was concentrated under reduced pressure to give compound D as a dihydrochloride salt.

Lithium 2-(3-amino-4-formylphenoxy)acetate (20 mg) and HBTU (38 mg) were placed in a 20 mL glass vial, and 2 mL anhydrous DMF was added. The resulting slurry was stirred at ambient temperature for 30 minutes, then compound D (38 mg) and DIEA (52 μl) were added. The reaction was stirred at ambient temperature for 17 hours. LCMS analysis confirmed that no starting material remained, but a bis-acylated by-product was formed with the desired product. The reaction mixture was diluted with EtOAc, washed _{successively with H 2} O and saturated aqueous NaCl, _{dried over Na 2} SO ₄ , filtered and concentrated under reduced pressure. The residue was taken up in 5 mL THF and treated with 1 mL aliquots of 1 M aqueous LiOH for 2 hours at ambient temperature, at which point LC-MS analysis showed that the bis-acylated product hydrolyzed to the desired product. Indicated that it has become. The reaction mixture was partitioned between EtOAc and water, the organic layer was separated, washed with saturated aqueous NaCl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude material was subjected to preparative RP-HPLC for purification. The HPLC eluent containing the desired product was diluted with EtOA, washed with saturated aqueous NaHCO ₃ and saturated aqueous NaCl to remove trifluoroacetic acid, _{dried over Na 2} SO ₄ , filtered and concentrated under reduced pressure, the title Compound N-(1-(3-amino-4-formylphenoxy)-2-oxo-7,10,13-trioxa-3-azahexadecane-16-yl)-4-((4-( 2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)methyl)benzamide (TU3627-042) was obtained.

Synthesis of 4-(2-(5-amino-8- methylbenzo[f][1,7]naphthyridin- 2-yl)ethyl)-3 -methylphenol (compound B)

1-ethynyl-4-methoxy-2-methylbenzene (commercially available) (1.1 eq), 3,5-dichloropicolinonitrile (1 eq), triethylamine (5 eq) in a round bottom flask capped with a septum , And anhydrous DMF (0.2 M) were added. Vacuum treatment and nitrogen flushing were performed 3 times. CuI (0.05 eq) and bis(triphenylphosphine)dichloro-palladium(II) (0.05 eq) were added. The diaphragm was replaced with a reflux condenser and the flask was heated at 60° C. overnight under a nitrogen atmosphere. When the reaction was completed by monitoring by TLC, the contents of the flask were loaded onto a large silica gel column pretreated with hexane. The product 3-chloro-5-((4-methoxy-2-methylphenyl)ethynyl)picolinonitrile was obtained by flash chromatography (silica gel, hexane:EtOAc (1:4%)).

To a round bottom flask equipped with a reflux condenser, 3-chloro-5-((4-methoxy-2-methylphenyl)ethynyl)picolinonitrile (from the previous step) (1 equivalent), tert-butyl 2-(4) ,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate (1.25 equivalents), K ₃ PO ₄ (2 equivalents), tris(dibenzylideneacetone) Dipalladium(0) (0.05 eq), and 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (0.1 eq) were added. n-butanol and water (5:2, 0.2 M) were added, and the contents were degassed 3 times (vacuum followed by nitrogen flushing). The reaction mixture was stirred vigorously overnight at 100° C. under nitrogen in an oil bath. The contents were cooled, dissolved in 200 mL of water, and extracted with methylene chloride. The combined organic layers were dried (Na ₂ SO ₄ ) and concentrated. Product 2-((4-methoxy-2-methylphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthy by flash chromatography (silica gel, 0-50% _{EtOAc in CH 2} Cl ₂₎ Lidin-5-amine was obtained.

In a round bottom flask with bar stirring, 2-((4-methoxy-2-methylphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine (from above step Of) (1 equivalent) was added. After ethanol and methylene chloride (1:2, 0.2 M) were added, palladium in carbon (activated powder, wet, 10% on carbon, 0.1 eq) was added. After vacuuming the contents, hydrogen flushing was performed 3 times. The reaction mixture was stirred vigorously overnight at room temperature under a hydrogen balloon. Thereafter, the reaction mixture was filtered through a pad of celite, and subsequently the pad of celite was washed with methylene chloride and EtOAc until no UV absorption occurred in the filtrate. The combined organic wash was concentrated. Product 2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridine- by flash chromatography (silica gel, 0-50% _{EtOAc in CH 2} Cl ₂₎ 5-amine was obtained.

_{CH 2} Cl in a stirred solution (0.2 M) of 2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine in methylene chloride in an ice water bath a 1 N solution of BBr ₃ (2 eq) of ₂ was added drop wise. The reaction was quenched with methanol within 30 minutes and concentrated in vacuo to give a crude residue. The crude was purified by flash chromatography on a COMBIFLASH® system (ISCO) using 0-20% methanol in dichloromethane, resulting in 4-(2-(5-amino-8-methylbenzo[f]). [1,7]naphthyridin-2-yl)ethyl)-3-methylphenol was obtained as a white solid.

Example 38-2: 4-((4-(2-(5-amino-8- methylbenzo[f][1,7]naphthyridin- 2-yl) ethyl)-3-methylphenoxy)methyl Synthesis of )-N-(1-(aminooxy)-2-oxo-7,10,13-trioxa-3-azahexadecane-16-yl)benzamide hydrochloride (TU3627-044).

HBTU (38 mg), 2-(tert-butoxycarbonylaminooxy)acetic acid (Fluka, 25 mg), DIEA (44 μL) and 5 mL DMF were combined in a 20 mL glass vial. The reaction was stirred at ambient temperature for 30 minutes, then compound D (38 mg) was added. The reaction was stirred at ambient temperature for 17 hours. LC-MS analysis confirmed that there was no starting material left, but several times overacylated by-products were formed with the desired product. The reaction mixture was diluted with EtOAc, washed _{successively with H 2} O and saturated aqueous NaCl, _{dried over Na 2} SO ₄ , filtered and concentrated under reduced pressure. The residue was taken up in 5 mL THF and treated with 1 mL aliquots of 1 M aqueous LiOH for 2 hours at ambient temperature, at which point LC-MS analysis showed that the hyperacylated product was hydrolyzed to the desired product. Indicated. The reaction mixture was partitioned between EtOAc and water, the organic layer was separated, washed with saturated aqueous NaCl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude was purified by silica gel flash chromatography using B in 5% solvent A (A: DMC; B: NH _{3 in 2% MeOH) to give the desired compound, E.}

Compound E (28 mg) was treated with 3 M methanolic HCl for 30 min at ambient temperature and concentrated under reduced pressure. This operation was repeated. Title compound 4-((4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)methyl)-N-( 1-(Aminooxy)-2-oxo-7,10,13-trioxa-3-azahexadecane-16-yl)benzamide hydrochloride (TU3627-044) was obtained as a yellow solid.

Example 38-3: N- (58- (3- amino-4-formyl-phenoxy) -15,55- dioxo-4,7,10,18,21,24,27,30,33,36 ,39,42,45,48,51-pentadecaoxa-14,54-diazaoctapentacontyl)-4-((4-(2-(5-amino-8-methylbenzo[f][1, Synthesis of 7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)methyl)benzamide (X3678-114).

A mixture of dPEG-acid F (100 mg), HATU (52.8 mg) and DIEA (97 μL) in 2.0 mL DMF was stirred at room temperature for 30 min. Amine D (prepared from 109 mg of compound C) was added and the reaction was stirred at room temperature until completion, monitored by HPLC. The product was isolated by preparative RP-HPLC. The Boc group of product G (138 mg) from the previous step was removed by treatment with 3 N HCl in methanol and then concentrated to dryness. A mixture of lithium 4-(3-amino-4-formylphenoxy)butanoate (25.2 mg), HATU (38.0 mg) and DIEA (69.7 μL) in 2.0 mL DMF was stirred at room temperature for 30 minutes. Amine H was added and the reaction was stirred at room temperature until monitored to be complete by HPLC. The product (X3678-114) was isolated by preparative HPLC.

Example 38-4: 2-(4-acetyl-3- aminophenoxy )-N-(3 -nitrobenzyl ) acetamide Synthesis of (TU633-068).

Lithium 2-(4-acetyl-3-aminophenoxy)acetate (215 mg), HBTU (379 mg), and 10 mL DMF are combined in a 20 mL glass vial and the resulting slurry is stirred at ambient temperature for 1 hour. And at this point the reaction mixture became almost homogeneous. Then 3'-nitrobenzylamine HCl (192 mg) and DIEA (191 μL) were added to the vial and the reaction was stirred at ambient temperature for 30 minutes. The reaction mixture was diluted with EtOAc, and water, saturated aq. Washed successively with NaCl, _{dried over Na 2} SO ₄ , filtered and concentrated under reduced pressure. The crude was purified by silica gel flash chromatography (hexane/EtOAc) to give the title compound as a yellow solid.

Example 38-5: Preparation of 2- (3-amino-4-formyl-phenoxy) -N- (4- nitrobenzyl) acetamide (TU3627-020).

Lithium 2-(3-amino-4-formylphenoxy)acetate (44.2 mg) and HBTU (79.6 mg) were placed in a 20 mL glass vial, and anhydrous DMF (5 mL) was added. The resulting slurry was stirred at ambient temperature and became homogeneous within 10 minutes. After 15 minutes, DIEA (50 μl) and 4′-nitrobenzylamine HCl (37.7 mg) were added to the reaction mixture and the reaction was stirred at ambient temperature. After 30 minutes, the reaction was confirmed to be complete by LC-MS analysis. The reaction mixture was partitioned between EtOAc and water. The organic layer was separated, and the light aq. Citric acid, saturated aq. NaHCO ₃ , and saturated aq. Washed successively with NaCl, _{dried over Na 2} SO ₄ , filtered and concentrated under reduced pressure to give the title compound as a tan solid.

Example 38-6: Preparation of 2- (3-amino-4-formyl-phenoxy) -N- (2- (2,4- dinitrophenyl) ethyl) acetamide By a similar method as in (TU3627-022).

^{Except for the use of N 1} -(2,4-dinitrophenyl)ethane-1,2-diamine (Oakwood, cat # 015083, 45.2 mg) instead of 4'-nitrobenzylamine HCl in the absence of DIEA Then, the title compound was prepared in the same manner as in TU3627-020. TU3627-022 was obtained as a yellow solid. Rf. 0.15 (SiO ₂ , MeOH in 5% DCM).

Example 38-7: 4- (3-amino-4-formyl-phenoxy) -N- (2- (2,4- dinitrophenyl) ethyl) Synthesis of butane amide (TU3627-088).

Lithium 4-(3-amino-4-formylphenoxy)butanoate (50 mg), HBTU (80 mg) and DMF (2 mL) were combined in a 20 mL glass vial and stirred at ambient temperature. After 30 minutes, N ¹ -(2,4-dinitrophenyl)ethane-1,2-diamine (oakwood, 45 mg) was added in one portion and the reaction was stirred at ambient temperature overnight. The reaction mixture was diluted with EtOAc and light aq. Citric acid, water, saturated aq. NaHCO ₃ , water, and saturated aq. Washed successively with NaCl, _{dried over Na 2} SO ₄ , filtered and concentrated under reduced pressure to give the title compound as a yellow solid. Rf: 0.18 (SiO ₂ , EtOAc).

Example 38-8: 4- (3-amino-4-formyl-phenoxy) -N- (4-nitrobenzyl) butane amide Synthesis of (3793-001).

HATU (114.1 mg) was added to a 1 mL DMF solution of lithium 4-(3-amino-4-formylphenoxy)-butanoate (68.7 mg) and the reaction was shaken at room temperature for 1 hour. The resulting solution was then added to a 1 mL DMF solution of 4-nitrobenzylamine HCl salt (56.6 mg) and triethylamine (84 μL), and another 1 mL DMF was allowed to aid the transfer. The reaction was stirred at room temperature for 2 hours. Upon completion, the reaction mixture was _{partitioned between 4 mL of 10% NaCl (aq)} and 8 mL of EtOAc. The phases were separated and the aqueous layer was extracted again with 8 mL EtOAc. The combined organic layers were _{dried over Na 2} SO ₄ , filtered and concentrated under reduced pressure to give a crude orange oil. The crude oil was purified by silica gel flash column chromatography with gradient elution of 0-10% MeOH/DCM to give the desired product as a light yellow solid.

Example 38-9: Synthesis of 3-(4-acetyl-3 -aminophenyl )-2-(4- nitrobenzamido )propanoic acid (X3471-116).

To 4-nitrobenzoic acid (50.1 mg), HATU (114 mg) and DIEA (105 μL) was added 1 mL DMF, and the mixture was stirred for 30 minutes. The resulting solution was then added to 3-(4-acetyl-3-aminophenyl)-2-aminopropanoic acid (66.7 mg, 1.0 eq) in 1.0 mL DMF and the reaction stirred at room temperature while monitoring by LC-MS. I did. The title product was then isolated by preparative HPLC.

Example 38-10: 4- (3-amino-4-formyl-phenoxy) butanoyl Synthesis of -AlaGlySerArgSerGly (D- Ala) LysChaValAlaAlaTrpThrLeuLysAla (D -Ala) Gly-OH (3647-104).

The Fmoc group of Fmoc-exPADRE CLEAR resin (549.3 mg, 0.1 mmol, purchased from Peptide International Inc.) was removed with 20% piperidine/DMF (8 mL x 3). The resin was washed with DMF (1.5 mL x 5). The resin was then lithium 4-(3-amino-4-formylphenoxy)-butanoate (34.4 mg), HBTU (45.5 mg), HOBt (16.2 mg), and 3 mL DMF of DIEA (52 μL). The solution was treated for 6 hours at room temperature. The resin was washed with DMF, and _{peptides were cleaved from the resin at room temperature for 2 hours using H 2} O/Me ₂ S/TFA (10/10/80 v/v%, 10 mL). The cut slurry was filtered through glass wool and most of the TFA was removed from the filtrate by evaporation. The residue was neutralized with 1 N NaOH _{( aq )} , diluted with acetonitrile, and dialyzed using a SpectraPro™ 7 dialysis membrane (MWCO 1000) in _{50% MeCN ( aq ) (4L x 10).} The solution remaining in the dialysis membrane was lyophilized to give the desired product as a white solid.

Example 38-11: Synthesis of 3-(4-acetyl-3 -aminophenyl ) propanoyl- Gly(D- Ala)LysChaValAlaAlaTrpThrLeuLysAla(D-Ala)Gly-OH (3465-143).

The Fmoc group of the Fmoc-PADRE CLEAR resin (105.3 mg, 0.02 mmol, purchased from Peptide International Inc.) was removed with 20% piperidine/DMF (8 mL x 3). The resin was washed with DMF (1.5 mL x 5). The resin was then coupled with a 0.5 mL DMF solution of lithium 3-(4-acetyl-3-aminophenyl)propanoate (5.1 mg), HBTU (9.1 mg), and HOBt (3.3 mg) at room temperature for 7 hours. Made it. The resin was washed with DMF and the _{peptide was cleaved from the resin for 2 hours using TIPS/H 2} O/TFA (5/55/90 v/v%, 3 mL). The cut slurry was filtered through glass wool and most of the TFA was removed from the filtrate by evaporation. The residue was washed with hexane (3 mL x 3 ₎ and dissolved in _{50% MeCN ( aq).} After lyophilization, a crude product was obtained, which was then purified by preparative HPLC to give the desired product as a light yellow powder.

Example 38-12: Preparation of 6- (4- (3-amino-4-formyl-phenoxy) butane amido) hexyl ^{-5'- * T * C * C *} A * T * G * A * C * G ^* T ^* T ^* C ^* C ^* T ^* G ^* A ^* C ^* G ^* T ^* T-3' (*: Phosphothioate) (3647-057) synthesis.

HBTU (6.1 mg), HOBt (2.2 mg), and triethylamine (7 μl) were added to a 1 mL DMSO solution of lithium 4-(3-amino-4-formylphenoxy)-butanoate (4.6 mg). Was added and the reaction was shaken at room temperature for 1 hour. Then, a 276 μl aliquot of the resulting solution was added to the amino-modified BG1 oligo (12.1 mg, 1.8 μmol, purchased from Integrated DNA Technologies, Inc.) and triethylamine (15). Μl) of 1.2 mL DMSO solution, and the reaction was shaken at room temperature for 2 days. The reaction mixture was diluted with water and dialyzed against water (4 L x 10) using Slide-A-Riser™ (MWCO 3500). The dialyzed solution was lyophilized to give the desired product as a white solid.

Example 38-13: 6-(3-(4-acetyl-3 -aminophenyl ) propanamido ) hexyl- 5'- ^* T ^* C ^* G ^* T ^* C ^* G ^* T ^* T ^* T ^* T ^{Synthesis of *} C ^* G ^* G ^* C ^* G ^* C ^* G ^* C ^* G ^* C ^* C ^* G-3' (*:phosphothioate) (3597-033).

HBTU (5.9 mg) and HOBt (2.1 mg) were added to a 1 mL DMSO solution of lithium 3-(4-acetyl-3-aminophenyl)propanoate (3.3 mg) and the reaction was shaken at room temperature for 1 hour. . Then, a 20 μl aliquot of the resulting solution was added to a 0.2 mL DMSO solution of amino-modified BG2 oligo (1.88 mg, 0.26 μmol, purchased from Integrated DNA Technologies, Inc.) and triethylamine (2 μl). Was added and the reaction was shaken at room temperature for 20 hours. Applying the resulting mixture to a NAP-25 ™ column equilibrated in H ₂ O, and eluted with H ₂ O. Every 1 mL fraction was monitored by LC-MS, the fractions containing the desired product were combined and lyophilized to give the desired product as a white solid.

Example 38-14: Preparation of 6- (4- (3-amino-4-upon-formyl phenoxy) butane amido) hexyl ^{-5'- * T * C * G *} T * C * G * T * T * T ^{Synthesis of *} T ^* C ^* G ^* G ^* C ^* G ^* C ^* G ^* C ^* G ^* C ^* C ^* G-3' (*:phosphothioate) (3597-167).

HATU (7.4 mg) was added to a 1 mL DMSO solution of lithium 4-(3-amino-4-formylphenoxy)-butanoate (5.5 mg) and the reaction was shaken at room temperature for 1 hour. Then, a 60 μl aliquot of the resulting solution was added to a 0.6 mL DMSO solution of amino-modified BG2 oligo (6.9 mg, 1.0 μmol) and triethylamine (7.5 μl), and the reaction was shaken at room temperature for 20 hours. . Then, another 60 μl aliquot of the activated ester solution freshly prepared in the same manner except for the use of HBTU instead of HATU was added to the reaction mixture, and the reaction was shaken for an additional 2 days. The reaction mixture was separated into 3 parts. It applied to a NAP-25 ™ column equilibrated in the respective portions of H ₂ O and eluted with H ₂ O. Every 1 mL fraction was monitored by LC-MS, the fractions containing the desired product were combined and lyophilized to give the desired product as a white solid.

Example 39: spin-cover- ABA Synthesis of reagents

Example 39-1: N- (3- amino-4-formylphenyl) -1-hydroxy-3-pyrroline-1-oxyl -2,2,5,5- tetramethyl-3-carboxamide Synthesis of (X3626-112b).

To 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid (55.3 mg), HBTU (102 mg) and DIEA (105 μL) were added 2.0 mL DMF. After stirring at room temperature for 30 minutes, 2,4-diaminobenzaldehyde (40.8 mg) was added. The mixture was stirred at room temperature until the reaction was complete, monitored by HPLC. The title product was isolated by preparative HPLC.

Example 40: biotin- ABA Synthesis of reagents

Example 40-1: N- (3- amino-4-formylphenyl) -1- (biotin amido) -3,6,9,12- tetrahydro-oxa-15-pentadecane amide (X3626-140) of synthesis.

1.0 mL DMF was added to 2,4-diaminobenzaldehyde (11.5 mg), NHS-dPEG™4 biotin (Quanta Biodesign, cat # 10200, 50 mg) and DMAP (10.4 mg). The reaction mixture was stirred at room temperature until the reaction was complete, monitored by HPLC. The product was isolated by silica gel flash chromatography.

Example 40-2: N- (1- (3- amino-4-formyl-phenoxy) -2-oxo -7,10,13- tree oxa-3-aza-hexadecane -16- yl) - Biotin amide Synthesis of (X3626-142).

To lithium 2-(3-amino-4-formylphenoxy)acetate (45.3 mg), HBTU (77.8 mg) and DIEA (31 μL) was added 1 mL DMF, and the mixture was stirred for 30 minutes. The resulting solution was added to Biotin-dPEG™ ₃ -NH ₃ ⁺ TFA (Quanta Biodesign, cat# 10193, 100 mg), DIEA (47 μL) in 1.0 mL DMF, and the reaction was stirred at room temperature while monitoring by LCMS. I did. The product was isolated by preparative HPLC.

Example 41: fluorescence- PEG - ABA Synthesis of reagents

Example 41-1: Fluorescein - PEG - ABA ( X3757 -48).

DMF (2.0 mL) was added to NHS-fluorescein A (23.6 mg), amine B (17.6 mg) and DIEA (8.7 μl). The mixture was stirred at room temperature until A was consumed as monitored by HPLC. Product C was isolated by preparative HPLC. ESI-MS (m/z) 679.72 (MH ⁺ ). Then, the product (C, 7.4 mg) was dissolved in 3 M HCl (1.0 mL), stirred at room temperature for 5 minutes, and then methanol was removed by evaporation. This operation was repeated to remove the Boc group to obtain amine D. To amine D lithium 4-(3-amino-4-formylphenoxy)butanoate (2.3 mg), HBTU (3.8 mg), DIEA (7.0 μL) and DMF (2 mL) were added at room temperature. The title product was then isolated by preparative HPLC.

Example 42: Oligosaccharides - ABA Synthesis of reagents

Example 42-1: of 3-amino-4- formylphenoxybutyrate Synthesis of Gal - Glu -1-amide (3793-050).

NaBH ₃ CN (94.3 mg) _{was added to a H 2 O (10 mL) solution (pH 7) of NH 4} OAc (771 mg) and lactose (180 mg), and the reaction was stirred at 35° C. for 7 days. The reaction mixture was lyophilized, then the residue was dissolved in H ₂ O, also Wexford (Dowex) 1X8-400 anion exchange resin was passed through a (OH- form, 45 g), an excess of BH ₃ CN ^-, its by-product And the acetate was removed. The eluent was lyophilized to give a crude product, which was purified by cation exchange chromatography using Dowex 50WX8-400 resin (H+ form, 30 g) to give the desired product (3793-048) as a light yellow powder. I did.

Lithium 3-amino-4-formylphenoxybutyrate (6.0 mg) was treated with HBTU (9.9 mg) and DIEA (7.7 μL) in anhydrous DMSO (200 μL) for 1 hour. Subsequently, a DMSO (250 μl) solution of Gal-Glu-1-amine (3793-048) (7.7 mg) from above was added to the reaction mixture at room temperature, followed by stirring for 1 day. The reaction mixture was lyophilized and _{purified by preparative HPLC-MS eluting with NH 4} OAc to give the desired product (3793-050) as a yellow powder.

Example 43: phospholipid- ABA Synthesis of reagents

Example 43-1: DOPE - ABA ( TU3627 -092).

Lithium 4-(3-amino-4-formylphenoxy)butanoate (34 mg) and HBTU (57 mg) were placed in a 20 mL glass vial, and 2 mL DMF was added. To activate, the reaction was stirred at ambient temperature for 30 minutes. DOPE (76 mg, 1,2,-dioleoyl-sn-glycero-phosphoethanolamine, NOF Corp.) was placed in a separate 20 mL vial, followed by DIEA (35 μL) and 3 mL DCM. The yellow solution of the activated ester in the first vial was transferred to the second vial and the reaction was stirred at ambient temperature. After 24 hours, the entire reaction mixture was applied to a 12 g pre-packed SiO _{2 column equilibrated with solvent A (solvent A: NEt 3} in 5% DMC, solvent B: NEt ₃ in 5% MeOH) and the column was zero → Elution over 15 minutes with a linear gradient of B in 15% A to give the partially purified product as a light yellow very viscous oil. This product was _{purified again by flash chromatography using a 12 g SiO 2} column (solvent A: NEt ₃ in 5% DMC, solvent B: NEt ₃ in 5% MeOH), linear gradient of B in 2 → 10% A And eluted over 15 minutes. Fractions containing pure product were concentrated under reduced pressure to give the triethylammonium salt of the title compound. Rf: 0.43 (SiO ₂ , MeOH in 10% DCM).

Example 44: PCL Reduction of coupling connection

It was found to reduce the PCL coupling linkage to prevent dissociation of the PCL-based protein conjugate. Figure 54A shows the ESI mass spectrometric analysis of hFGF21-Lys150PCL after coupling to 2-ABA and _{reduced with 20 mM NaCNBH 3 for 1 hour.} Figure 54B shows the ESI mass spectrometric analysis of the reduced hFGF21-Lys150PCL 2-ABA conjugate after dialysis with 10 mM phosphate buffer (pH 7.5) and incubation for 1 day at 50°C.

Figure 55 shows the stability of PCL ligation to PEGylated FGF21, reduced or not reduced by _{NaCNBH 3.} The FGF21 mutant Arg154PCL was reacted with 30.3 kDa-2-ABA-PEG (TU3627-024; see Example 37-7) and purified. Purified FGF21Arg154PCL-30.3 kDa-2-ABA-PEG _{was reduced with 20 mM NaBH 3} CN for 16 hours (room temperature, pH 7.5, 100 mM protein). Samples were incubated for 5 minutes at 4° C., room temperature, 37° C. and 50° C., and 95° C. for 16 hours. SDS-PAGE gels of the reduced and non-reduced samples are shown in Figure 55A. Additionally, Figure 55B shows SDS-PAGE gels for unreduced samples incubated at 4° C., room temperature, 37° C. and 50° C., and 95° C. for 60 hours.

Example 45: NMR study of PCL- A ( Lys - P5C ) and PCL- B (Lys-P2C) covalently modified by 2-ABA.

Reaction of PCL-A (3647-125, see Example 36-1) and PCL-B (3793-011, see Example 36-2) with 2-aminobenzaldehyde (2-ABA) and addition of the resulting PCL-ABA The structure of water was studied by standard 1D and 2D nuclear magnetic resonance (NMR) spectroscopy.

For the reaction of PCL-A and PCL-B with 2-ABA and subsequent characterization of its products, ^{Bruker Avance equipped with a 1} H/ ¹³ C/ ¹⁹ F/ ³¹ P-QNP-low temperature probe NMR data were obtained at 300 K on a 400 MHz NMR instrument (Bruker Biospin, Billerica, MA). ¹ H 1D spectra were typically recorded with 16 scans, relaxation delay of 5 s, and 16384 complex data points with a search width of 12 ppm. ^{The 1} H- ¹ H COSY spectrum was typically recorded as a 4 scan, 256 t ₁ experiment, and the ¹ H- ¹ H ROESY spectrum was recorded as an 8 scan, 512 t _{1 experiment.} ^{The 1} H- ¹³ C HMBC spectrum is typically recorded as a 32 scan, 256 t ₁ experiment, and the ¹ H- ¹³ C HMQC spectrum is typically a 4 scan, 128 t ₁ experiment, 222 ppm on the carbon scale and 12 ppm on the proton scale Or 7.5 ppm of spectral width.

To characterize the reduced adduct, all spectra were recorded at 300 K on a Bruker Avans 600 MHz instrument equipped with a ¹ H/ ¹³ C/ ^{15 N-TXI-low temperature probe. A 1} H spectrum was typically recorded with 64 scans, relaxation delay 2 s, and 16384 complex data points excitation for water molecule suppression, with a search width of 14 ppm. ^{The 1} H- ¹ H COSY spectrum was typically _{recorded with a 16 scan, 1024 t 1} experiment using a spectral width of 10 ppm. ^{The 1} H- ¹³ C HMQC spectrum was typically recorded in 8 scans, 256 t ₁ experiments with a spectral width of 160 ppm on the carbon scale and 10 ppm on the proton scale. ¹ H- ¹³ C HMBC spectra were typically recorded with 88 scans, 256 t ₁ experiments at 300 K, using a spectral width of 180 ppm on the carbon scale and 10 ppm on the proton scale.

The reaction of PCL-A and 2-ABA was monitored as follows: 1.0 mg of PCL-A synthesized as described in Example 36-1 was dissolved in 0.5 mL of 1X PBS _{in D 2 O.} 10 μl of 10 mM 3-(trimethylsilyl)propionic acid (TSP) in D _{2 O was added as an internal standard and to determine the concentration by NMR.} 3.7 mg of 2-aminobenzaldehyde (2-ABA; purchased from Sigma) was dissolved in 0.5 mL of 1×PBS _{in D 2 O and 10 μL of 10 mM TSP.} The concentration of both samples was measured by NMR. NMR signals of the starting materials (Table 7) were determined using standard NMR methods including ¹ H 1D, ¹ H- ¹ H COSY, ¹ H- ¹ H ROESY, ¹ H- ¹³ C HMBC and ¹ H- ^{13 C HMQC experiments.} Assigned.

To initiate the reaction, 325 μl of PCL-A solution was mixed with 175 μl of 2-ABA solution. The resulting reaction mixture was transferred to an NMR tube and contained PCL-A and 2-ABA in an approximate 1:1 molar ratio. The reaction was allowed to proceed at room temperature, and the NMR spectrum was periodically obtained at the time point indicated (FIG. 56). The reaction signal for the starting PCL-A material (dot) and 2-ABA reactant (asterisk) disappeared quickly and while the reaction proceeded to completion, all PCL-A was converted (there was a slight excess of 2-ABA in the sample. I did). At the first time point obtained after 0.5 hours of mixing, two new species were detected in a ratio of approximately 2:1 (representative resonances are indicated by arrows). A few species have been completely converted to major species after several days have elapsed.

The final reaction product of PCL-A and 2-ABA was ^{standard 1D 1} H and ¹³ C, 2D ¹ H- ¹ H COSY, ¹ H- ¹ H ROESY, ¹ _{using samples prepared in D 2} O and d6-DMSO. It was characterized by H- ¹³ C HMBC and ¹ H- ¹³ C HMQC NMR spectroscopy. Samples in d6-DMSO were purified by HPLC. The signal assignment in the case of proton and carbon resonance and ^{the correlations observed in the 1} H- ¹³ C HMBC spectrum in d6-DMSO are summarized in Table 8.

Figure 57A shows the correlation of atom numbering and selected bond passage for HMBC. In contrast to that observed with the PCL-A starting material, two methylene protons on carbon 17 were observed at two different chemical shift values. These protons also showed a heteronuclear bond pass-through correlation for carbon 7 in the HMBC spectrum, suggesting that a covalent bond was formed between nitrogen 16 and carbon 7. The protons on carbon 7 resonate at 5.6 ppm (proton 7 is a signal of the PCL-A/2-ABA adduct highlighted in FIG. 201) and correlate for carbons 2, 8, 9, 5 and 13. Similarly, protons on carbon 2 showed an HMBC correlation for carbon 9. These and all other NMR observations for the two samples characterized in D ₂ O and D6-DMSO are consistent with the structure in Figure 57A depicted for the main product of the PCL-A/2-ABA adduct. do. Thus, overall NMR evidence indicates that the structure of the reaction product between PCL-A and 2-ABA is as follows:

In addition, it was attempted to clarify the structural characteristics of the hydrophobic form observed in the reaction mixture (FIG. 56). The low concentration of the minority form and the fact that it slowly converts to the main form complicates the analysis. The analysis did not come to a conclusion. Both minority and major forms have one proton on carbon 7 and the chemical shifts of both were very similar (indicated by arrows at 5.6 ppm in FIG. 56). In addition, the space-passing ROESY contact was similar in both forms. The two methylene protons on carbon 17 were degenerated, meaning that they exhibited the same chemical shift in the case of unreacted PCL-A as well as the minor form of the PCL-A/2-ABA adduct. However, the chemical shifts of these protons are distinguished in their main form. In addition, these protons show an HMBC correlation for carbon 7 in both minor and major forms, suggesting a covalent bond between carbon 7 and nitrogen 16. In the hydrophobic form, it was observed that the chemical shift of the methylene proton on carbon 17 was degraded, which means that the covalent bond between carbon 7 and nitrogen 16 may be semi-stable, and the NMR observations for the hydrophobic form showed that chemical exchange between two or more species. It can be implied that it is the result of The minority form may be an alternative semi-stable stereoisomer of the main form in chemical exchange with the protonated form of the PCL-A/2-ABA adduct (FIG. 57B ).

In addition, _{a solution of synthetic PCL-A and 2-ABA in D 2} O was mixed as described above and monitored by NMR to allow the reaction to proceed and complete. The PCL-A/2-ABA adduct was reduced to a new species by adding an aliquot of a solution of sodium cyanoborohydride (NaCNBH _{3 ) in D 2 O to the NMR tube. Additional 3} aliquots of NaCNBH were added until the resonance at 5.6 ppm characteristic for the PCL-A/2-ABA adduct (FIG. 56, arrows) disappeared completely. Then, the NMR sample of the fully reduced PCL-A/2-ABA adduct was repeatedly lyophilized and _{redissolved in anhydrous D 2} O to reconcentrate the sample and remove water. The final sample was reconstituted in 0.5 mL of anhydrous D _{2 O.} The reduced form was characterized by standard 2D ¹ H- ¹ H COSY, ¹ H- ¹³ C HMBC and ¹ H- ¹³ C HMQC NMR spectroscopy. ^{The signal assignment of protons and carbon resonances from the 1} H- ¹³ C HMBC spectrum and the heteronuclear binding pass-through correlations are summarized in Table 9.

The numbering of the atoms is shown in Figure 57C. The key differences in the correlations observed in the major forms of PCL-A/2-ABA adducts are the lack of heteronuclear bond pass-through correlations between the methylene protons on carbon 17 and carbon 7, and between the protons on carbon 2 and carbon 9. Is that there is no correlation through the bond. These and all other NMR observations are consistent with the following (also in Figure 57C) structure for the reduced PCL-A/2-ABA adduct:

In addition, the reaction of PCL-B and 2-ABA was studied under the same conditions as those for reacting PCL-A and 2-ABA. PCL-B synthesized as described in Example 36-2 was _{dissolved in D 2} O (as described above), and a reaction mixture containing PCL-B and 2-ABA in a molar ratio of approximately 1:1 was NMR. Transferred to the tube. The signal assignments of the starting materials are listed in Table 10.

The reaction was allowed to proceed at room temperature, and NMR spectra were obtained periodically at the indicated time points (FIG. 58). In contrast to the PCL-A sample, PCL-B did not react easily with 2-ABA. Even after 17 days, a large amount of starting material (asterisk for 2-ABA; dots for PCL-B) still remained, and only a small amount was converted to a new species (arrow). This species could not be further characterized. However, NMR analysis of the two reactions clearly showed that the reactivity of PCL-A with 2-ABA was much greater than that of PCL-B.

Example 46: mEGF Pyrrolysine incorporated in ( Pyl ) And PCL of Derivatization

Incorporation of pyrrolysine (Pyl) and PCL into mEGF was achieved as described in Example 14, E. E. coli BL21(DE3) cells were cotransformed with the mutant mEGF Tyr10TAG gene on pAra-pylSTBCD and pET22b vector. The resulting mEGF mutant is hereinafter referred to as mEGF-Tyr10PCL/Pyl. ESI-MS analysis revealed that both PCL and PYL were incorporated at position 10 of mEGF (FIG. 59A; expected mass for mEGF Tyr10PCL = 7296 Da; expected mass for mEGF Tyr10Pyl = 7310 Da). Accordingly, a mixture of mEGF proteins into which PCL or PYL was incorporated was obtained. In this particular sample, the predominant species was mEGF with PCL incorporated therein.

To demonstrate the derivatization of this mixture and to demonstrate that PYL was modified by the method provided herein, mEGF Tyr10PCL/ The ABA reagent TU3627-014 (see Example 34-2) was coupled to PYL. The conjugation reaction was initiated by adding 10 μM mEGF Tyr10PCL/PYL and 1 mM TU3627-014. Formation of the protein conjugate was confirmed by electrospray ionization-mass spectrometry (ESI-MS) (Fig. 59B). The ratio between PCL and PYL adducts was similar to the ratio of PCL and PYL in the unreacted protein (FIG. 58A ), indicating that the reactivity to PCL and Pyl is similar.

It is understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes will be suggested to those skilled in the art in that respect, which are included within the spirit and scope of the present application and the scope of the appended claims. .

SEQUENCE LISTING <110> IRM Geierstanger, Bernhard <120> BIOSYNTHETICALLY GENERATED PYRROLINE-CARBOXY-LYSINE AND SITE SPECIFIC PROTEIN MODIFICATIONS VIA CHEMICAL DERIVATIZATION OF PYRROLINE-CARBOXY-LYSINE AND PYRROLYSINE RESIDUES <130> PAT053160-WO <160> 35 <170> PatentIn version 3.5 <210> 1 <211> 1365 <212> DNA <213> Methanosarcina mazeii <400> 1 atggataaaa aaccactaaa cactctgata tctgcaaccg ggctctggat gtccaggacc 60 ggaacaattc ataaaataaa acaccacgaa gtctctcgaa gcaaaatcta tattgaaatg 120 gcatgcggag accaccttgt tgtaaacaac tccaggagca gcaggactgc aagagcgctc 180 aggcaccaca aatacaggaa gacctgcaaa cgctgcaggg tttcggatga ggatctcaat 240 aagttcctca caaaggcaaa cgaagaccag acaagcgtaa aagtcaaggt cgtttctgcc 300 cctaccagaa cgaaaaaggc aatgccaaaa tccgttgcga gagccccgaa acctcttgag 360 aatacagaag cggcacaggc tcaaccttct ggatctaaat tttcacctgc gataccggtt 420 tccacccaag agtcagtttc tgtcccggca tctgtttcaa catcaatatc aagcatttct 480 acaggagcaa ctgcatccgc actggtaaaa gggaatacga accccattac atccatgtct 540 gcccctgttc aggcaagtgc ccccgcactt acgaagagcc agactgacag gcttgaagtc 600 ctgttaaacc caaaagatga gatttccctg aattccggca agcctttcag ggagcttgag 660 tccgaattgc tctctcgcag aaaaaaagac ctgcagcaga tctacgcgga agaaagggag 720 aattatctgg ggaaactcga gcgtgaaatt accaggttct ttgtggacag gggttttctg 780 gaaataaaat ccccgatcct gatccctctt gagtatatcg aaaggatggg cattgataat 840 gataccgaac tttcaaaaca gatcttcagg gttgacaaga acttctgcct gagacccatg 900 cttgctccaa acctttacaa ctacctgcgc aagcttgaca gggccctgcc tgatccaata 960 aaaatttttg aaataggccc atgctacaga aaagagtccg acggcaaaga acacctcgaa 1020 gagtttacca tgctgaactt ctgccagatg ggatcgggat gcacacggga aaatcttgaa 1080 agcataatta cggacttcct gaaccacctg ggaattgatt tcaagatcgt aggcgattcc 1140 tgcatggtct atggggatac ccttgatgta atgcacggag acctggaact ttcctctgca 1200 gtagtcggac ccataccgct tgaccgggaa tggggtattg ataaaccctg gataggggca 1260 ggtttcgggc tcgaacgcct tctaaaggtt aaacacgact ttaaaaatat caagagagct 1320 gcaaggtccg agtcttacta taacgggatt tctaccaacc tgtaa 1365 <210> 2 <211> 1050 <212> DNA <213> Methanosarcina mazeii <400> 2 atgatccaga aaatggcaac cgaagaactt gacaggttcg gggagaaaat tattgaaggt 60 tttaaattgt ctgatgatga cctcagggct cttctttctc ttgaattcga agaagagctg 120 gaaaagcttt actatgtagc tagaaaggtc agaaactatt atttcggcaa cagggtgttt 180 cttaactgtt ttatttattt ctcaacttat tgtaaaaacc agtgctcttt ttgctactat 240 aactgtaaaa acgaaattaa ccgctaccgc ctgaccggtg aagaggttaa agagatgtgc 300 aaagccctga aaggtgcagg ctttcacatg atcgacctga caatgggaga ggatccctat 360 tactatgatg accctgaccg cttcgttgaa cttgtcagga cagtaaaaga agaactcggg 420 cttccaataa tgatttctcc gggagttatg gatgacagca ccctcctgaa agccagggaa 480 gaaggagcaa atttctttgc cctttatcag gagacttatg accgcgaact ttatggaaag 540 ctaagggtag gtcagtcctt cgaaggaagg tttaatgccc gcaggtttgc aaaagaacag 600 gggtactgta tagaagacgg cattcttacc ggcgtaggaa atgatatcga atcaactctt 660 atatccctga aggggatgaa agcaaacaat cctgatatgg taagggtaat gacttttctg 720 cctcaggaag gaactccgct tgaaggtttc agcgatagtt caaagctttc ggagctgaaa 780 atcatagcga ttctcaggct catgtttcct gaatgcctga taccggcttc tcttgacctt 840 gaaggcatag acggcatggt gcaccgttta aatgccggag caaatattgt aacctccatc 900 ctcccagatt cacgcctgga aggggttgcc aattacgacc gcggcatgga agagagggac 960 agggacgtta caagcgttgt caaaaggctg aaggttatgg gaatggaacc tgcgccgcag 1020 gctgagtttg agagagtcct ggggtgctaa 1050 <210> 3 <211> 1116 <212> DNA <213> Methanosarcina mazeii <400> 3 atgagagagt cctggggtgc tagcctgaaa acaatatgcc ttataggcgg gaagctgcag 60 ggcttcgagg ctgcatacct atctaagaaa gccggaatga aagtgcttgt aatagacaaa 120 aacccgcagg cgcttataag gaattatgcg gatgagttcc agtgttttaa cataacggaa 180 gagccggaaa aactcgtcgc gatatcaaaa aatgttgatg ccatactgcc ggtaaatgaa 240 aaccttgaat gtatagaatt tctgaattct ataaaagaaa aattctcctg cccggtactt 300 ttcgattttg aagcttacag gatcagcagg gataagagaa aatcaaaaga atacttcgca 360 tccataggaa ccccgacccc tcaggacaaa ccgtcggaac caccttattt tgtaaagcct 420 ccctgcgaaa gcagcagtgt gggagcgaga ataatccatg acaggaaaga gcttaaagag 480 cttgagcccg ggatgctcat agaagaatac gttgaagggg aagtggtctc acttgaggtc 540 ataggggatg gaaataattt tgctgtggta aaggaaaccc ttgtacatat cgatgacacc 600 tatgactgcc atatggtgac ccctctccct ctagaccctt ccttcaggga actatcctac 660 tcccttgcag caaacctgcc cttaaaagga attatggacg tggaagcgat ttccggcccc 720 ctggggttaa aagttattga gatagatgcc cgtttcccga gccagactcc gactgcggtc 780 tattattctt ccgggatcaa cctcatagaa ctcctgttcc gggcttttaa tggaggcata 840 gaagagatca aaactctccc tgaagacagg tactgcattt acgaacatct catgcttgca 900 gaaaatggag tacttatccc tgtgggagaa caggtcctgt ccatgggaaa tgattacggc 960 aattattatg aagaacctgg aatagagatt ttcctgtgca aaggagagaa ccctgtattc 1020 accctggttt tctggggcag agacagggaa gaagctgaag ctagaaaaaa caaagggctt 1080 tcgattctaa aaagccgttt cggagctgct gcataa 1116 <210> 4 <211> 795 <212> DNA <213> Methanosarcina mazeii <400> 4 atggcacttt taaccccaga agacctggaa aatattaaca aacagcttca agaagctgat 60 tctactgtcc gcagagttac agggcttgat ataaaaggta tctgtaaaga tttctacggc 120 acaactccat gctgtgaaaa agtaggtatc gtgcctgtga cctcagggaa cgggatcata 180 gggagctttt ccgaatccct gaatgcaatt gccgggtatt tcgggtttga cagttttatt 240 actgatatgc ctgacgtcag cggatattat gaggcagtaa agaacggagc ccggatcata 300 cttatggcag atgataatac cttccttgcc cacaacctga aaaatggaaa aatcgccaat 360 aaccagccgt gtacaggcat aatttatgct gaaatagctt caagatacct gaaagccgat 420 tccaaagaag tgcttgccgt gggtcttggg aaggttggat ttccgggagc agcccatctc 480 gtacagaaag gcttcaaggt ttacggatat gatgctgaca gaacccttct agaaaaaagc 540 gtttccagcc tcggaattat acctttcaat cccgtcagcc ccgaaggcga caggcaaagg 600 aagttttcca ttattttcga agcaaccccc tgtgcagaca cgattccgga atccgtaatt 660 tcggaaaact gtgtgatttc tacccctggg ataccctgtg caatctcaaa ggagctgcaa 720 aaaaagtgtg gagttgaact tgtaatggaa ccactgggga taggtacagc atcaatgctg 780 tattctgtac tctaa 795 <210> 5 <211> 72 <212> DNA <213> Methanosarcina mazeii <400> 5 ggaaacctga tcatgtagat cgaatggact ctaaatccgt tcagccgggt tagattcccg 60 gggtttccgc ca 72 <210> 6 <211> 223 <212> PRT <213> Homo sapiens <400> 6 Met Lys Thr Phe Ile Leu Leu Leu Trp Val Leu Leu Leu Trp Val Ile 1 5 10 15 Phe Leu Leu Pro Gly Ala Thr Ala Gln Pro Glu Arg Asp Cys Arg Val 20 25 30 Ser Ser Phe Arg Val Lys Glu Asn Phe Asp Lys Ala Arg Phe Ser Gly 35 40 45 Thr Trp Tyr Ala Met Ala Lys Lys Asp Pro Glu Gly Leu Phe Leu Gln 50 55 60 Asp Asn Ile Val Ala Glu Phe Ser Val Asp Glu Thr Gly Gln Met Ser 65 70 75 80 Ala Thr Ala Lys Gly Arg Val Arg Leu Leu Asn Asn Trp Asp Val Cys 85 90 95 Ala Asp Met Val Gly Thr Phe Thr Asp Thr Glu Asp Pro Ala Lys Phe 100 105 110 Lys Met Lys Tyr Trp Gly Val Ala Ser Phe Leu Gln Lys Gly Asn Asp 115 120 125 Asp His Trp Ile Val Asp Thr Asp Tyr Asp Thr Tyr Ala Val Gln Tyr 130 135 140 Ser Cys Arg Leu Leu Asn Leu Asp Gly Thr Cys Ala Asp Ser Tyr Ser 145 150 155 160 Phe Val Phe Ser Arg Asp Pro Asn Gly Leu Pro Pro Glu Ala Gln Lys 165 170 175 Ile Val Arg Gln Arg Gln Glu Glu Leu Cys Leu Ala Arg Gln Tyr Arg 180 185 190 Leu Ile Val His Asn Gly Tyr Cys Asp Gly Arg Ser Glu Arg Asn Leu 195 200 205 Leu Asp Tyr Lys Asp Asp Asp Asp Lys His His His His His His 210 215 220 <210> 7 <211> 672 <212> DNA <213> Homo sapiens <400> 7 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 8 <211> 672 <212> DNA <213> Homo sapiens <400> 8 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg taggccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 9 <211> 672 <212> DNA <213> Homo sapiens <400> 9 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctagctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 10 <211> 672 <212> DNA <213> Homo sapiens <400> 10 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactagg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 11 <211> 672 <212> DNA <213> Homo sapiens <400> 11 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtagtg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 12 <211> 672 <212> DNA <213> Homo sapiens <400> 12 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtacta gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 13 <211> 672 <212> DNA <213> Homo sapiens <400> 13 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctagctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 14 <211> 672 <212> DNA <213> Homo sapiens <400> 14 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtag 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 15 <211> 672 <212> DNA <213> Homo sapiens <400> 15 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tagaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 16 <211> 672 <212> DNA <213> Homo sapiens <400> 16 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttagtgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 17 <211> 10 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (7)..(7) <223> Xaa is a PCL residue <400> 17 Tyr Trp Gly Val Ala Ser Xaa Leu Gln Lys 1 5 10 <210> 18 <211> 29 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (7)..(7) <223> Xaa is Cyc residue <400> 18 Lys Asp Pro Glu Gly Leu Xaa Leu Gln Asp Asn Ile Val Ala Glu Phe 1 5 10 15 Ser Val Asp Glu Thr Gly Gln Met Ser Ala Thr Ala Lys 20 25 <210> 19 <211> 166 <212> PRT <213> Homo sapiens <400> 19 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp Arg 165 <210> 20 <211> 166 <212> PRT <213> mouse <400> 20 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Val Thr Met Gly Cys Ala Glu Gly 20 25 30 Pro Arg Leu Ser Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg Met Glu Val Glu Glu Gln Ala Ile Glu Val Trp 50 55 60 Gln Gly Leu Ser Leu Leu Ser Glu Ala Ile Leu Gln Ala Gln Ala Leu 65 70 75 80 Leu Ala Asn Ser Ser Gln Pro Pro Glu Thr Leu Gln Leu His Ile Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Ser Leu Leu Arg Val Leu 100 105 110 Gly Ala Gln Lys Glu Leu Met Ser Pro Pro Asp Thr Thr Pro Pro Ala 115 120 125 Pro Leu Arg Thr Leu Thr Val Asp Thr Phe Cys Lys Leu Phe Arg Val 130 135 140 Tyr Ala Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Val 145 150 155 160 Cys Arg Arg Gly Asp Arg 165 <210> 21 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 21 Met Gly Asp Ser Lys Ile His His His His His His Glu Asn Leu Tyr 1 5 10 15 Phe Gln Gly <210> 22 <211> 301 <212> PRT <213> Homo sapiens <400> 22 Met Gly Ser Asp Lys Ile His His His His His His Glu Asn Leu Tyr 1 5 10 15 Phe Gln Gly Ser Leu Leu Val Asn Pro Glu Gly Pro Thr Leu Met Arg 20 25 30 Leu Asn Ser Val Gln Ser Ser Glu Arg Pro Leu Phe Leu Val His Pro 35 40 45 Ile Glu Gly Ser Thr Thr Val Phe His Ser Leu Ala Ser Arg Leu Ser 50 55 60 Ile Pro Thr Tyr Gly Leu Gln Cys Thr Arg Ala Ala Pro Leu Asp Ser 65 70 75 80 Ile His Ser Leu Ala Ala Tyr Tyr Ile Asp Cys Ile Arg Gln Val Gln 85 90 95 Pro Glu Gly Pro Tyr Arg Val Ala Gly Tyr Ser Tyr Gly Ala Cys Val 100 105 110 Ala Phe Glu Met Cys Ser Gln Leu Gln Ala Gln Gln Ser Pro Ala Pro 115 120 125 Thr His Asn Ser Leu Phe Leu Phe Asp Gly Ser Pro Thr Tyr Val Leu 130 135 140 Ala Tyr Thr Gln Ser Tyr Arg Ala Lys Leu Thr Pro Gly Ser Glu Ala 145 150 155 160 Glu Ala Glu Thr Glu Ala Ile Cys Phe Phe Val Gln Gln Phe Thr Asp 165 170 175 Met Glu His Asn Arg Val Leu Glu Ala Leu Leu Pro Leu Lys Gly Leu 180 185 190 Glu Glu Arg Val Ala Ala Ala Val Asp Leu Ile Ile Lys Ser His Gln 195 200 205 Gly Leu Asp Arg Gln Glu Leu Ser Phe Ala Ala Arg Ser Phe Tyr Tyr 210 215 220 Lys Leu Arg Ala Ala Glu Gln Tyr Thr Pro Lys Ala Lys Tyr His Gly 225 230 235 240 Asn Val Met Leu Leu Arg Ala Lys Thr Gly Gly Ala Tyr Gly Glu Asp 245 250 255 Leu Gly Ala Asp Tyr Asn Leu Ser Gln Val Cys Asp Gly Lys Val Ser 260 265 270 Val His Val Ile Glu Gly Asp His Arg Thr Leu Leu Glu Gly Ser Gly 275 280 285 Leu Glu Ser Ile Ile Ser Ile Ile His Ser Ser Leu Ala 290 295 300 <210> 23 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 23 Met Gly Ser Ser His His His His His His Leu Glu Val Leu Phe Gln 1 5 10 15 Gly Pro <210> 24 <211> 125 <212> PRT <213> Homo sapiens <400> 24 Met Gly Ser Ser His His His His His His Leu Glu Val Leu Phe Gln 1 5 10 15 Gly Pro Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr 20 25 30 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu 35 40 45 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe 50 55 60 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 65 70 75 80 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro 85 90 95 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His 100 105 110 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu 115 120 125 <210> 25 <211> 196 <212> PRT <213> Homo sapiens <400> 25 Met Gly Ser Ser His His His His His His Ser Ser Gly Glu Asn Leu 1 5 10 15 Tyr Phe Gln Gly Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Val Arg 20 25 30 Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu 35 40 45 Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro 50 55 60 Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile 65 70 75 80 Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala 85 90 95 Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu 100 105 110 Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly 115 120 125 Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala 130 135 140 Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala 145 150 155 160 Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly 165 170 175 Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro 180 185 190 Ser Tyr Ala Ser 195 <210> 26 <211> 173 <212> PRT <213> mouse <400> 26 Met Arg Gly Ser His His His His His His His Gly Ser Gly Ile Glu Gly 1 5 10 15 Arg Leu Arg Ser Ser Ser Gln Asn Ser Ser Asp Lys Pro Val Ala His 20 25 30 Val Val Ala Asn His Gln Val Glu Glu Gln Leu Trp Leu Ser Gln Arg 35 40 45 Ala Asn Ala Leu Leu Ala Asn Gly Met Asp Leu Lys Asp Asn Gln Leu 50 55 60 Val Val Pro Ala Asp Gly Leu Tyr Leu Val Tyr Ser Gln Val Leu Phe 65 70 75 80 Lys Gly Gln Gly Cys Pro Asp Tyr Val Leu Leu Thr His Thr Val Ser 85 90 95 Arg Phe Ala Ile Ser Tyr Gln Glu Lys Val Asn Leu Leu Ser Ala Val 100 105 110 Lys Ser Pro Cys Pro Lys Asp Thr Pro Glu Gly Ala Glu Leu Lys Pro 115 120 125 Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly 130 135 140 Asp Gln Leu Ser Ala Glu Val Asn Leu Pro Lys Tyr Leu Asp Phe Ala 145 150 155 160 Glu Ala Ser Gly Gln Val Tyr Phe Gly Val Ile Ala Leu 165 170 <210> 27 <211> 62 <212> PRT <213> mouse <400> 27 Met Asn Ser Tyr Pro Gly Cys Pro Ser Ser Tyr Asp Gly Tyr Cys Leu 1 5 10 15 Asn Gly Gly Val Cys Met His Ile Glu Ser Leu Asp Ser Tyr Thr Cys 20 25 30 Asn Cys Val Ile Gly Tyr Ser Gly Asp Arg Cys Gln Thr Arg Asp Leu 35 40 45 Arg Trp Trp Glu Leu Arg Leu Glu His His His His His His 50 55 60 <210> 28 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <220> <221> MOD_RES <222> (2)..(2) <223> D-alanine <220> <221> MOD_RES <222> (4)..(4) <223> cyclohexyl-alanine <220> <221> MOD_RES <222> (4)..(4) <223> cyclohexyl-alanine <220> <221> MOD_RES <222> (13)..(13) <223> D-alanine <400> 28 Gly Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Xaa Gly 1 5 10 <210> 29 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <220> <221> MOD_RES <222> (7)..(7) <223> D-alanine <220> <221> MOD_RES <222> (9)..(9) <223> cyclohexyl-alanine <220> <221> MOD_RES <222> (9)..(9) <223> cyclohexyl-alanine <220> <221> MOD_RES <222> (18)..(18) <223> D-alanine <400> 29 Ala Gly Ser Arg Ser Gly Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys 1 5 10 15 Ala Xaa Gly <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic construct <220> <221> misc_feature <222> (1)..(20) <223> phosphothioate backbone <400> 30 tccatgacgt tcctgacgtt 20 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> synthetic construct <220> <221> misc_feature <222> (1)..(22) <223> phosphothioate backbone <400> 31 tcgtcgtttt cggcgcgcgc cg 22 <210> 32 <211> 22 <212> PRT <213> mouse <220> <221> MOD_RES <222> (11)..(11) <223> PCL <400> 32 Met Asn Ser Tyr Pro Gly Cys Pro Ser Ser Xaa Asp Gly Tyr Cys Leu 1 5 10 15 Asn Gly Gly Val Cys Met 20 <210> 33 <211> 22 <212> PRT <213> mouse <220> <221> MISC_FEATURE <222> (11)..(11) <223> pyrrolysine <400> 33 Met Asn Ser Tyr Pro Gly Cys Pro Ser Ser Xaa Asp Gly Tyr Cys Leu 1 5 10 15 Asn Gly Gly Val Cys Met 20 <210> 34 <211> 14 <212> PRT <213> mouse <220> <221> MISC_FEATURE <222> (3)..(3) <223> pyrrolysine <400> 34 Asn His Xaa Val Glu Glu Gln Leu Glu Trp Leu Ser Gln Arg 1 5 10 <210> 35 <211> 14 <212> PRT <213> mouse <220> <221> MOD_RES <222> (3)..(3) <223> PCL <400> 35 Asn His Xaa Val Glu Glu Gln Leu Glu Trp Leu Ser Gln Arg 1 5 10

Claims (12)

&Lt; / RTI >
(I)

(Wherein,
R ₁ is H or an amino terminal modification group;
R ₂ is OH or a carboxy terminus modification group;
n is an integer from 1 to 5000;
Each AA is independently selected from an amino acid residue, a pyrrolysine amino acid residue, a pyrrolysine analog amino acid residue having the structure of Formula A-1, and a pyrrolysine analog amino acid residue having the structure of Formula B-1, ;

Wherein R ₆ is H or C ₁ alkyl
At least one AA is a pyrrolysine analog amino acid residue having the structure of Formula A-1 or Formula B-1, the amino acid residue of Formula A-1 is a residue of the amino acid of Formula V, the amino acid residue of Formula B-1 Is a residue of an amino acid of formula

Wherein R ₆ is H or C ₁ alkyl
Amino acids of formula (V) or formula (VI) are biosynthetically produced in cells in contact with a growth medium comprising a pylC gene and a pylD gene and comprising a precursor) The compound of claim 1, wherein R ₁ is H, the amino acid of formula V is an amino acid having the structure of formula VII, the amino acid of formula VI is an amino acid having the structure of formula VIII, and the precursor is D-ornithine, D Arginine, (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid or (2S) -2-amino-6-((R) -2,5-diaminopentaneami Fig. 8) Hexanoic acid compound.

The compound of claim 1, wherein R ₁ is C ₁ alkyl, the amino acid of formula V is an amino acid having the structure of formula IX, and the precursor is D-ornithine, D-arginine, (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid or (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid;
Amino acid of formula VI is an amino acid having a structure of formula X, precursor is D-ornithine, D-arginine, 2,5-diamino-3-methylpentanoic acid, (2R, 3S) -2,5-dia Mino-3-methylpentanoic acid, (2R, 3R) -2,5-diamino-3-methylpentanoic acid, (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid Or (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid.

The cell of claim 1, wherein the cells further comprise a pylS gene and a pylT gene, tRNA and aminoacyl tRNA synthetase, wherein the aminoacyl tRNA synthetase is the gene product of the pylS gene, and tRNA Is a gene product of the pylT gene. The cell of claim 1, wherein the cell further comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS), wherein the O-RS represents the O-tRNA. A compound for aminoacylating an amino acid of Formula V, Formula VI, Formula VII, Formula VIII, Formula IX or Formula X. The compound of any one of claims 1-3, wherein the cells are prokaryotic or eukaryotic cells. The method of claim 6, wherein the cells are Escherichia coli ) cells, mammalian cells, yeast cells, insect cells, CHO cells, HeLa cells, HEK293F cells or sf9 cells. A method of derivatizing a protein comprising contacting a protein having a structure according to Formula I with a reagent of Formula III or Formula IV below.
(I)

(Wherein,
R ₁ is H or an amino terminal modification group;
R ₂ is OH or a carboxy terminus modification group;
n is an integer from 1 to 5000;
Each AA is independently selected from an amino acid residue, a pyrrolysine amino acid residue, a pyrrolysine analog amino acid residue having the structure of Formula A-1, and a pyrrolysine analog amino acid residue having the structure of Formula B-1, ;

R ₆ is H or C ₁ alkyl;
At least one AA is a pyrrolysine amino acid residue or a pyrrolysine analog amino acid residue having a structure of Formula A-1 or Formula B-1)
(III)

(IV)

(Wherein,
R _3, R ₅ and each R ₄ is H, -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX ¹ ;
A is C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ heterocycloalkyl, 5 to 6 membered monocyclic aryl, 5 to 6 membered monocyclic heteroaryl, fused 9 to 10 membered bicyclic ring or fused 13 a to 14-membered tricyclic ring, where A is -OH, -NO _2, halo, C _{1 - 8} alkyl, halo-substituted -C _{1 - 8} alkyl, hydroxy-substituted -C _{1 - 8} alkyl, Optionally substituted with 1 to 5 substituents independently selected from aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX ¹ ;
L is a bond, C _{1 - 8} alkylene, halo-substituted -C _{1 - 8} alkylene, hydroxy-substituted -C _{1 - 8} alkylene, C _2-8 alkenylene group, a halo-substituted -C _{2 - 8} alkenylene, hydroxy-substituted ₂ -C ₈ alkenylene,

-O (CR ¹¹ R ¹² ) _k- , -S (CR ¹¹ R ¹² ) _k- , -S (O) _k (CR ¹¹ R ¹² ) _k- , -O (CR ¹¹ R ¹² ) _k -NR ¹¹ C (O)-, -O (CR ¹¹ R ¹² ) _k C (O) NR ^11- , -C (O)-, -C (O) (CR ¹¹ R ¹² ) _k- , -C (S)-, -C (S) (CR ¹¹ R ¹² ) _k- , -C (O) NR ^11- , -NR ¹¹ C (O)-, -NR ¹¹ (CR ¹¹ R ¹² ) _k- , -CONR ¹¹ (CR ¹¹ R ¹² ) _k- , -N (R ¹¹ ) CO (CR ¹¹ R ¹² ) _k- , -C (O) NR ¹¹ (CR ¹¹ R ¹² ) _k- , -NR ¹¹ C (O) (CR ¹¹ R ¹² ) _k - is selected from wherein each R ¹¹ and R ¹² are independently H, C _{1 - 8} alkyl, and, _{- 8} alkyl, halo-substituted -C _{1 - 8} alkyl or hydroxy-substituted -C ₁ k is an integer from 1 to 12;
X ¹ is a label, dye, polymer, water soluble polymer, polyalkylene glycol, poly (ethylene glycol), derivative of poly (ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Labels, reactive compounds; Resins, peptides, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, PCR probes, antisense polynucleotides, ribo-oligonucleotides, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labels , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or noncovalently interact with other molecules, photocatalytic moieties, chemiradioactive excitation moieties, ligands, photoisomerization moieties, biotin, biotin analogues Residues containing heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox Active agents, aminothio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chromophore groups, chemiluminescent groups, fluorescent moieties, electron dense groups, magnetic groups, intercalating groups, chelating groups, chromophores , Energy transfer agents, biologically active agents, detectable labels, small molecules, inhibitory ribonucleic acids, siRNAs, radionucleotides, neutron-trapping agents, derivatives of biotin, quantum dot (s), nanotransmitters, radiotransmitters, abzymes, enzymes , Activated complex activators, viruses, toxins, adjuvants, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, phospholipids, LPS-like molecules, keyhole limpet hemoshi Non- (KLH), immunogenic hapten, aglycan, allergen, angiostatin, anti-hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecule, mimotope, receptor, reverse micelle, detergent, immune booster , brother Dye, FRET reagent, radiation-imaging probe, different probe spectroscopy, prodrugs, toxins for immunotherapy, a solid _{support, -CH 2 CH 2 - (OCH} 2 CH 2 O) p -OX 2, -O- (CH 2 _{CH 2 O) p CH 2 CH} 2 -X 2 , and are selected from any combination thereof, wherein p is 1 to 10,000, X ² is H, C _{1 - 8} alkyl, or a protecting group being the terminal functional group). The compound of claim 8, wherein ring A is furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, Pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, dithiane, thiomorpholine, pyridazine, pyrimidine, pyrazine , Piperazine, triazine, trithiane, indoliazine, indole, isoindole, indolin, benzofuran, benzothiophene, indazole, benzimidazole, benzthiazole, purine, quinolyzine, quinoline, isoquinoline, Cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine, quinuclidin, carbazole, acridine, phenazine, pentazine, phenoxazine, phenyl, indene, naphthalene, azulene, flu Orene, anthracene, phenanthracene, norborane and adamantin. 10. The method of claim 9, wherein ring A is selected from phenyl, naphthalene and pyridine. The method of claim 8, wherein the reagent of Formula IV is

/ RTI &gt;
(Wherein L and X ¹ are the same as described in claim 8) The method of claim 8, wherein the reagent of Formula IV is

Wherein exPADRE is a peptide according to SEQ ID NO: 28; PADRE is a peptide according to SEQ ID NO: 29; BG1 is a polynucleotide according to SEQ ID NO: 30; BG2 is a polynucleotide according to SEQ ID NO: 31.

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